Methods And Compositions For Targeted Imaging

ABSTRACT

A new approach to targeting imaging agents to macrophage-rich sites of interest is disclosed. Compositions of the invention are rHDL and HDL-like liposomal compositions, protein constituents of which, apolipoproteins A-I and/or A-II or fragments thereof are used not only as structural but also as targeting agents. This is achieved by certain controlled chemical or enzymatic modification of apolipoproteins A-I or A-II or fragments thereof. Such modification converts these apolipoproteins to substrates for macrophage scavenger receptors and results in the improvement of contrast agent-(HDL/modified apolipoprotein)-particle association with macrophages and/or absorption (uptake) by macrophages when compared to that of the contrast agent-(HDL/apolipoprotein)-particle constructed with non-modified naturally occurring apo A-I. The compositions can be used for noninvasive specific in vivo molecular detection and localization of macrophage-rich sites of interest using imaging techniques such as computed tomography (CT), gamma-scintigraphy, positron emission tomography (PET), single photon emission computed tomography (SPECT), magnetic resonance imaging (MRI).

CROSS-REFERENCE TO A RELATED APPLICATION

The present application is a Continuation and claims priority to U.S.non-provisional application Ser. No. 13/501,085, filed Apr. 9, 2012,entitled “Methods and Compositions for Targeted Imaging”. Thisapplication is a National Stage entry of International Application No.PCT/US2010/052117 filed on Oct. 10, 2010, which claims priority to andthe benefit of U.S. provisional application Ser. No. 61/250,465, filedOct. 9, 2009. The entire content of the aforementioned provisionalapplication is incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS_WEB

The official copy of the sequence listing is submitted electronicallyvie EFS-Web as an ASCII formatted Sequence Listing with a file named“2012_10_16_SBK-002_ST25.txt”, created on Oct. 16, 2012 and having asize of 1 kilobyte. The sequence listing contained in this ASCIIformatted document is part of the specification and is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a composition comprising a metallic ornon-metallic contrast agent covalently or noncovalently conjugated tolipoprotein nanoparticles wherein said nanoparticles comprise at leastone modified apolipoprotein and at least one lipid. The inventionfurther relates to the use of these compositions in imaging techniquessuch as computed tomography (CT), gamma-scintigraphy, positron emissiontomography (PET), single photon emission computed tomography (SPECT),magnetic resonance imaging (MRI), and combined imaging techniques.

BACKGROUND OF THE INVENTION 1. Cancer and Macrophage Imaging

Cancer is one of the major causes of mortality in the United States, andthe worldwide incidence of cancer continues to increase. At present,noninvasive imaging approaches, including x-ray-based computer-assistedtomography (CT), positron emission tomography (PET), single-photonemission tomography, and magnetic resonance imaging (MRI), are used asimportant tools for detection of human cancer (Wang et al. C A Cancer JClin 2008; 58:97-110). However, in vivo studies have shown that only 1to 10 parts per 100,000 of intravenously administered mAbs, therapeutic,or imaging agents can reach their parenchymal targets. Thus, greatertargeting selectivity and better delivery efficiency are the 2 majorgoals in the development of imaging contrast formulations. Thedevelopment of tumor-targeted contrast agents based on a nanoparticleformulation may offer enhanced sensitivity and specificity for in vivotumor imaging using currently available clinical imaging modalities.

Cancer cells secrete a variety of chemoattractants that attractmacrophages and cause them to accumulate in the tumor tissue wherein themacrophage becomes a tumor-associated macrophage (TAM) (Beckmann et al.WIREs Nanomed Nanobiotech 2009; 1:272-98). TAMs have a range offunctions with the capacity to affect diverse aspects of neoplastictissues including angiogenesis and vascularisation, stroma formation anddissolution, and modulation of tumor cell growth (enhancement andinhibition). These macrophages of M2 phenotype promote tumor cellproliferation and metastasis by secreting a wide range of growth andproangiogenic factors as well as metalloproteinases and by theirinvolvement in signaling circuits that regulate the function offibroblasts in the tumor stroma. The prognosis associated with TAMs isdependent on tumor type, but in breast cancer and prostate cancer, TAMaccumulation has been linked to decreased survival. This fact obviouslybrings an important perspective for macrophage tracking as a potentialdiagnostic tool in cancer.

One molecular imaging strategy to improve the specificity of cancerdetection is target-specific imaging of TAMs. The macrophage image ofregions of the subject's body at cancer risk can be used to assessmacrophage density and displacement associated with any primary canceror metastatic cancer in the subject, such density and displacement beingindicative of neoplasia. This image also can be used to identify thesite of biopsy in the subject, macrophage density being an indicator oftumor growth. For example, whole body MRI scanning and cancer stagingusing ultrasmall superparamagnetic iron oxide (USPIO) particles asmacrophage-seeking MRI agents to perform macrophage-enhanced MRI hasbeen suggested (US Pat Appl 20090004113).

In sum, the prior art teaches the use of contrast agents that are notspecific to cancer at all, namely gadolinium chelates and manganesecompounds, or contrast agents including perfluorocarbon compounds andbiofunctionalized nanoparticles containing perfluorocarbons andgadolinium for imaging arterial plaques and atherosclerotic vessels. Theclinical application of USPIO particles in cancer imaging (US Pat Appl20090004113) is limited: (i) iron oxide particles induce signal loss,making differentiation between iron-laden macrophages and imagingartifacts challenging (Hyafil et al. Arterioscler Thromb Vasc Biol 2006;26:176-81); (ii) two MRI studies are required before and after infusionof contrast medium; and, finally, (iii) the uptake of iron oxideparticles seems to be nonspecific, which may limit their use for cancerimaging.

2. Atherosclerotic Plaques and their Role in the Thromboembolic Events

Atherosclerosis remains the leading cause of death in industrializedsocieties, including the US. It accounts for half of the morbidity andmortality in Western countries, and incidence of atherosclerosis isprojected to increase worldwide in the next 2 decades (Michaud et al.Jama 2001; 285:535-39). It represents a systemic disease affecting thevessel walls of all the major arteries, including the aorta, coronary,carotid, and peripheral arteries, and leads to a myriad of diseases,including stroke, myocardial infarction, peripheral vascular disease,aortic aneurysms, and sudden death (Ross, R. N Engl J Med 1999;340:115-26). Accurate in vivo tracking of progressive lesions would beextremely useful clinically to determine the status of patients'atherosclerotic disease. In addition, accurate identification ofatherosclerotic wall mass, rather than the degree of lumen narrowing, isneeded to better understand the factors that result in plaqueprogression and regression, and to precisely determine the effectivenessof potential interventions such as aggressive lipid-lowering therapy.

A vast majority of the thromboembolic events result from rupture orerosion of atherosclerotic plaques prone to rupture in the coronaryarteries, so-called “high-risk” or “vulnerable” plaques (Shah, P. K.Prog Cardiovasc Dis 2002; 44:357-68; Virmani et al. Arterioscler ThrombVasc Biol 2000; 20:1262-75), which is characterized by further thinningand rupture of the thin fibrous cap (about 65-150 micron) overlying thethrombogenic large lipid core (Falk, E. Circulation 1992; 86:III30-42;Davies, M. J. & Thomas, A. C. Br Heart J 1985; 53:363-73). Thecharacteristics of high-risk or vulnerable plaques vary depending on thearterial region (i.e., coronaries, carotids, or aorta) in which they arefound. By using molecular probes that contain contrast-producingelements and specifically and sensitively bind or target differentmolecular and functional components of atherosclerotic plaque, molecularimaging enables imaging and identification high-risk and vulnerableplaques. By targeting appropriate components in the cascade ofatherosclerosis pathogenesis, this approach makes possible to preciselydiscern plaque constitution as well as stage/classify plaques. Thus,these molecularly targeted contrast agents/probes will be and are ableto detect features indicative of instability or vulnerability ofatheromatous plaques.

3. Non-MRI Techniques Used to Identify the Atherosclerotic Plaques

Different techniques have been used to target specific components ormolecules of atheromatous plaque (Choudhury, R. P. & Fisher, E. A.Arterioscler Thromb Vasc Biol 2009; 29:983-91; Jaffer et al.Arterioscler Thromb Vasc Biol 2009; 29:1017-24; Rudd et al. ArteriosclerThromb Vasc Biol 2009; 29:1009-16; Sosnovik, D. E. & Weissleder, R. CurrOpin Biotechnol 2007; 18:4-10; Desai, M. Y. & Bluemke, D. A. Magn ResonImaging Clin N Am 2005; 13:171-80, vii). Perfusion imaging, Doppler flowimaging studies, and angiography help detect luminal narrowing but notthe presence of inflamed and vulnerable atherosclerotic plaques innonstenotic vessels (Sosnovik, D. E. Radiology 2009; 251:309-10). X-rayangiography is a frequently used imaging modality to diagnose coronaryartery diseases and assess their severity. Traditionally, thisassessment is performed directly from the angiograms, and thus, cansuffer from viewpoint orientation dependence and from lack of precisionof quantitative measures due to magnification factor uncertainty. Usingof three dimensional (3D) reconstruction of the coronary arteries fromthe angiograms can lead to higher accuracy and reproducibility in thediagnosis and to better precision in the quantification of the severityof the diseases (Blondel et al. Phys Med Biol 2004; 49:2197-208). Still,because a major limitation of X-ray angiography is being a “luminogram,”alternative imaging modalities to detect atherosclerotic plaque areneeded and have been developed. Intravascular ultrasound is acatheter-based technique which produces tomographic two-dimensionalcross-sectional images of vessel wall architecture and plaque(Fitzgerald et al. Circulation 1992; 86:154-8) and allows to discernplaque components accurately (Nissen, S. E. and Yock, P. Circulation2001; 103:604-16), but it is an invasive procedure and is associatedwith procedure-related complications. In addition, the ability ofintravascular ultrasound to image the vessel wall downstream from astenosis is limited. Furthermore, because of its high cost,intravascular ultrasound is not suitable for screening purposes in anasymptomatic population. Accuracy of B-mode ultrasonography that canalso be used to measure plaque volume in the carotid arteries is limitedby the plane of acquisition and the fact that atherosclerosis is a focalprocess (O'Leary, D. H. & Polak, J. F. Am J Cardiol 2002; 90:18L-21L;Spence, J. D. Am J Cardiol 2002; 89:10B-15B; discussion 15B-16B).Computer tomography (CT), including its powerful modification, theelectron beam tomography and multidetector computed tomography (Leber etal J Am Coll Cardiol 2004; 43:1241-7), is one of the major imagingmodalities that allows to evaluate patients with heart disease anddetect and quantify coronary calcification, but its ability to detectsoft, noncalcified plaques is not yet fully determined (Fayad et al.Circulation 2002; 106:2026-34).

4. Identification of the Atherosclerotic Plaques Using the MRI Technique

MRI is a non-invasive diagnostic technique that has the potential toimage some events at the cellular or subcellular level (Sosnovik, D. E.& Weissleder, R. Curr Opin Biotechnol 2007; 18:4-10). This technology isbased on the interaction of protons with each other and with surroundingmolecules in a tissue of interest. MRI produces images by measuring theresonance frequency (RF) signals arising from the magnetic moments oflipid and mainly water protons in living tissues (Strijkers et al.Anticancer Agents Med Chem 2007; 7:291-305; Haacke et al., De Graaf, R.A. In vivo NMR spectroscopy, Principles and Techniques. John Wiley &Sons: Chichester, N.Y., 1998).

The normal contrast in the MR images depends mainly on the proton spindensity and the longitudinal (T1) and transverse (T2 and T2*) relaxationtimes. There are many pathological conditions that do not lead tosignificant morphological changes and do not display specific enoughchanges in the relaxation times. Under those circumstances the pathologymay be detected using an MRI contrast agent that locally changes therelaxation times of the diseased tissue. The advantages of the use ofcontrast agents are considerable, although the use of contrast agentsviolates the non-invasive character of MRI to some extent. Thecombination of MRI and contrast agents greatly enhances thepossibilities to depict inflamed tissues like in arthritis (Lutz et al.Radiology 2004; 233:149-57), tumor angiogenesis (Collins, D. J. &Padhani, A. R. IEEE Eng Med Biol Mag 2004; 23:65-83), atheroscleroticplaques (Rudd et al. Arterioscler Thromb Vasc Biol 2009; 29:1009-16;Sanz, J. & Fayad, Z. A. Nature 2008; 451:953-7), and the break down ofthe blood brain barrier related to pathologies such as multiplesclerosis (Veldhuis et al. J Cereb Blood Flow Metab 2003; 23:1060-9).

MRI offers several advantages over other imaging modalities: 1) it isnon-ionizing as it detects the magnetic signals generated by protons andother molecules; 2) the technique is tomographic, enabling anytomographic plane through a three-dimensional volume to be imaged; 3)high-resolution images with excellent soft tissue contrast betweendifferent tissues can be obtained; 4) multiple contrast mechanisms arepossible using MRI, and 5) the technique can be used to provideanatomical as well as physiological readouts. Because of its highresolution, 3D capabilities, noninvasive nature, and capacity for softtissue characterization, is emerging as a powerful modality to assessthe atherosclerotic plaque burden in the arterial wall and has been usedto monitor atherosclerosis in vivo (Yuan et al. Circulation 1998;98:2666-71; Amirbekian et al. Proc Natl Acad Sci USA 2007; 104:961-6; USPat Appl 20070243136). MRI allows high-resolution imaging of thearterial wall without ionizing radiation (Rudd et al. ArteriosclerThromb Vasc Biol 2009; 29:1009-16). Spatial resolutions of 250 micronare possible for aorta (Yonemura et al. J Am Coll Cardiol 2005;45:733-42) and carotid plaque (Yuan et al. Circulation 1998;98:2666-71). MRI can image the extent of atherosclerosis and monitor theefficacy of antiatherosclerotic treatments (Fayad et al. Circulation2000; 101:2503-9; Corti et al. Circulation 2002; 106:2884-7). Inaddition, elements of the mature atherosclerotic plaque (fibrous cap,lipid core, hemorrhage) can be identified using MRI (Kerwin et al. TopMagn Reson Imaging 2007; 18:371-8). However, imaging inflammation withinatherosclerotic plaque using MRI requires the injection of a contrastagent.

5. MRI Contrast Agents

The developments in recent years in the field of cellular and molecularimaging have boosted the search for new and better MRI contrast agents.Cellular and molecular imaging aim to visualize molecular and cellularprocesses non-invasively in vivo (Choy et al. Mol Imaging 2003;2:303-12; Delikatny, E. J. & Poptani, H. Radiol Clin North Am 2005;43:205-20; Frias et al. Contrast Media Mol Imaging 2007; 2:16-23). Thisis achieved by directing a detectable reporter, e.g. a nuclear tracer oran MRI contrast agent, towards the target molecules or processes ofinterest.

Today, magnetic resonance (MR) contrast media (or contrast agents) areused in 40-50% of all MR examinations worldwide and the degree ofcontrast utilization is expected to increase in the future (Bellin, M.F. Eur J Radiol 2006; 60:314-23; Bellin, M. F. & Van Der Molen, A. J.Eur J Radiol 2008; 66:160-7). MR contrast media are administered toenhance tissue contrast, to characterize lesions and to evaluateperfusion and flow-related abnormalities. They include non-specificextracellular contrast agents and organ-specific contrast agents, mostlyliver specific contrast agents. In 2007, of the 27.5 million MRIprocedures performed in the U.S., 43% used a contrast agent as part ofthe imaging procedure.

MR contrast agents are diagnostic pharmaceutical compounds containingsuperparamagnetic or paramagnetic metal ions that affect the MR-signalproperties of surrounding tissues. Superparamagnetic contrast agentsshorten the transverse magnetization (T2*) and induce MR signal loss onT2*-weighted sequences (“negative” contrast). These agents are based oniron oxide particles and can be classified according to particle size.Microparticles of iron oxide (MPIO) are largest, followed bysuperparamagnetic iron oxides (SPIO), and finally USPIO. Iron-ladenmacrophages can be detected in the aortic subendothelium, and the effectof cytokine injection upon cell infiltration can be studied. Following aretrospective clinical study (Schmitz et al. J Magn Reson Imaging 2001;14:355-61), this work has recently translated into a prospective patienttrial (Kooi et al. Circulation 2003; 107:2453-8), where it was foundthat uptake occurred mainly in ruptured and rupture-prone plaques andnot in stable lesions, suggesting that the two can be differentiated inorder to assess the relative risk for stroke and embolic complications.However, iron oxide-based T2 contrast agents have several disadvantageswhich limit their use in MRI imaging: a) the ambiguity of the signalvoid which is a general disadvantage of negative contrast imaging; b)the contrast generated by the labeled cells is limited if backgroundsignal is low; c) negative contrast on T2-weighted scans, which can benonspecific and difficult to distinguish from other causes of signalhypointensity (such as calcification, susceptibility artifacts,flow-related signal loss, or air) and thus make image interpretationsubjective; d) the correlation between iron oxide concentration and T2contrast is not always linear; and finally, e) heavy loading canincrease transverse relaxivity (R2), disproportionate to the amount ofiron present per image voxel (compartmentalized iron oxide can cause amore substantial reduction in local relaxation time thannon-compartmentalized iron oxide), which complicates quantitativeinterpretation of the results (Medarova, Z. & Moore, A. Nat RevEndocrinol 2009; 5:444-52). In addition, because of the disadvantageouslarge T2*/T1 ratio, USPIO compounds are less suitable for arterial boluscontrast enhanced MR angiography than paramagnetic gadolinium complexes.Paramagnetic Gd3+ (gadolinium; a member of the lanthanide group ofelements) ion represents the stable ion with seven unpaired electrons,the largest number of unpaired electrons that are paramagnetic. Gd-basedcontrast agents (GBCAs) enhance the longitudinal magnetization (T1) ofnearby water protons resulting, in contrast to superparamagneticcontrast agents, in a positive signal on the MR image.

6. Use of Iron Oxide Particles for Imaging of Atherosclerosis

Iron oxide particles have been used for imaging of atherosclerosis(Amirbekian et al. Proc Natl Acad Sci USA 2007; 104:961-6). Macrophageuptake of iron oxide nanoparticles involves macrophage scavengerreceptor (MSR)-mediated endocytosis and depends mainly on the size ofcontrast agents (Raynal et al. Invest Radiol 2004; 39:56-63). However,there are factors that limit the clinical application of this approach:(i) iron oxide particles induce signal loss, making differentiationbetween iron-laden macrophages and imaging artifacts challenging (ii)due to limited plaque permeation, high doses (several times the clinicaldose) and long delay times (up to 5 days post-injection) are required;and finally, (iii) the uptake of iron oxide particles seems to benonspecific, which may limit their use for plaque imaging.

7. Gadolinium-Containing MRI Contrast Agents

All available GBCAs are chelates that contain the gadolinium ionGd3+(Bellin, M. F. & Van Der Molen, A. J. Eur J Radiol 2008; 66:160-7).Free gadolinium is highly toxic. Chelation of gadolinium by appropriateligands dramatically reduces its acute toxicity. Gadolinium chelates arethe most widely used extracellular, non-specific contrast agents.Approximately 30% of 20 million MR imaging scans that are performed onlyin the US annually, use GBCAs, and therefore approximately 6 milliondoses of GBCAs are administered annually (Kuo, P. H. J Am Coll Radiol2008; 5:29-35).

Nine intravenous GBCAs have been approved for clinical use in the USand/or international market: Magnevist® (gadopentetate dimeglumine;Bayer Shering Pharma), Dotarem® (gadoterate meglumine; Guerbet,Aulnay-sous-bois, France), Omniscan® (gadodiamide; Nycomed, Oslo,Norway), ProHance® (gadoteridol; Bracco SpA, Milan, Italy), Gadovist®(gadobutrol; Bayer Shering Pharma), MultiHance® (gadobenate dimeglumine;Bracco SpA), OptiMARK® (gadoversetamide; Mallinkrodt, St. Louis, USA),Primovist® (gadoxetic acid; Bayer Shering Pharma) in Europe, or Eovist®in USA, and Vasovist® (gadofosveset trisodium; Epix Pharmaceuticals,Cambridge, USA). To these figures one must add the administration ofagents approved outside the US: Dotarem, Gadovist, Vasovist andPrimovist. As of 2007, Magnevist was the leading MRI contrast agent inthe US and worldwide. Since its introduction, Magnevist has been used inover 80 million procedures worldwide and continues to be the moststudied MRI contrast agent on the market.

8. Gadolinium-Induced Nephrogenic Systemic Fibrosis

Nephrogenic systemic fibrosis (NSF) is a severe delayed fibroticreaction of the body tissues to GBCAs. NSF is a rare systemic disorderfirst described in 1997 which affects patients with chronic kidneydisease. Within weeks, it may lead to disability by formation ofcontractures. It may also lead to death. Since its recognition, therehave been more than 200 cases reported worldwide. The disease isexclusively seen in patients with various degree of renal failure andmost of whom have been exposed to GBCAs. The ratio of Gd to calcium intissue deposits correlates positively with the gadodiamide (Omniscan)dose and with serum ionized calcium at the time of Gd exposure. To date,the disease mechanism is still unclear and there is no proven treatmentfor NSF.

In 2007, NSF emerged as a major adverse consequence of gadoliniumchelate injection, although primarily involving weaker chelates ofgadolinium that were later approved (Khurana et al. Invest Radiol 2007;42:139-45). The first case was filed in 2007 by the mother of a patientwho died three years earlier after receiving a Magnevist injection aspart of an MRI procedure. As of February 2009, 241 U.S. lawsuitsinvolving Magnevist, which is the member of GBCA family, were pendingand it was anticipated that more would be lodged.

Currently, there is no cure for NSF and there are no alternativess forGd-based MRI contrast agents. Moreover, in angiographic studies, forexample, GBCAs have been suggested as a safer alternative toradiocontrast media (Ruangkanchanasetr et al. J Ren Care 2009; 35:11-5)that are known to cause acute renal failure (Solomon, R. Semin Nephrol1998; 18:551-7). Furthermore, the lowest possible dose of gadoliniumshould be used because development of NSF might be dose-related (Broomeet al. Am J Roentgenol 2007; 188:586-92).

All currently approved agents use the same basic principle for clinicalutility in MR scanning. Gd3+ ion is chelated for safety whilemaintaining its ability to provide enhancement on T1-weighted imaging(Kuo, P. H. J Am Coll Radiol 2008; 5:29-35). The chemical differences inthe chelates, which were previously of little clinical relevance, nowhave become very important in light of their potential differences inpropensity to cause NSF because free gadolinium is hypothesized toinduce NSF.

9. Targeted Delivery of Gadolinium-Containing MRI Contrast Agents

It is important to note that gadolinium chelates, currently the onlyclinically approved imaging agents in cardiovascular MRI, distributepassively to the extracellular space and do not reflect the degree ofactive inflammation, as acute and chronic infarction enhance alike(Fuster V. & Kim R. J. Circulation 2005; 112:135-44). In order toachieve good resolution of the MR image, a certain quantity of theimaging agent must accumulate at the site of interest being examined (USPat Appl 20070243136). Preferably, the imaging agent should specificallyaccumulate at the site being examined. For example, the required tissueconcentration of an MR contrast agent is about 10⁻⁴-10⁻⁶ M (Aime et al.J Magn Reson Imaging 2002; 16:394-406). For radionuclide imaging it isonly about 10⁻¹⁰ M. This is a great challenge since the molecularepitopes expressed at 10⁻⁹ or 10-01² molar concentrations must bedetected. Another challenge is to get the imaging agent to and into thesite of interest. The lower efficacy of the GBCAs, relative to ironoxides, necessitates the need for high paramagnetic payloads at the siteof interest. One way to reach the required local concentration of an MRcontrast agent is to increase the intravenously injected dose. However,for GBCAs this leads to higher risk of NSF and other adverse outcomes.Alternative and very promising approach is molecular MRI that entailsdelivering MRI contrast agents to locations of interest using moleculartargeting techniques and relies on the use of contrast agents thattarget specific cells or molecular pathways of relevance to disease. Inmolecular MRI, targeted carriers with the high affinity toward specificmolecular epitopes are used to deliver the imaging agents to the site ofinterest. Importantly, as the specificity of the delivery vehicle towardthe target increases, the portion of the injected GBCAs that isdelivered directly to the site of interest increases as well. Thisallows for a significant reduction of the overall systemic dose of thecontrast agent and, in case of GBCAs, diminishes the risk of NSF withoutcompromising the MRI quality.

Currently, contrast agents for tracking potentially important componentsof atherosclerotic disease are at various stages of development (Sanz,J. & Fayad, Z. A. Nature 2008; 451:953-7). Most of the available probesare in experimental testing, although some have already advanced toclinical evaluation. By producing GBCA-containing carriers specific tocomponents of atherosclerotic plaque, targeted imaging ofathersoclerosis helps to detect vulnerable (unstable) lesions prone toatherothrombotic effects (Frias et al. Contrast Media Mol Imaging 2007;2:16-23).

10. Atherosclerotic Plague Instability Correlates with its MacrophageContent

Inflammation has a crucial role at all stages of atherosclerosis. Forthis reason, macrophages are key in the progression of atherosclerosis,entering the intima as monocytes and being activated to macrophages viainteraction with and uptake of modified low density lipoprotein untilthey become foam cells and eventually forming the necrotic lipid coreassociated with unstable plaques (Lusis, A. J. Nature 2000; 407:233-41).There is a known link between a high and active macrophage content ofatherosclerotic plague and plaque instability (Hansson, G. K. N Engl JMed 2005; 352:1685-95). Furthermore, in humans, high macrophage contentin plaques is characteristic of vulnerability to rupture, which is theproximal cause of acute coronary syndromes (Amirbekian et al. Proc NatlAcad Sci USA 2007; 104:961-6). Unstable (symptomatic) carotid arteryplaques have been demonstrated to contain significantly higher number oflipid-laden macrophages than the stable (asymptomatic) ones (385+/−622vs. 1,114+/−1,104, P value<0.009) (Wakhloo et al. J Vasc Interv Radiol2004; 15:S111-21). Thus, the macrophage count that correlates with theprogression and prognosis of human atherosclerosis in general, and theatherosclerotic plaques in particular, can be used as a distinctivefeature of unstable plaques for molecular imaging purposes. It should benoted that because activated macrophages are the reliable indicators ofnot only atherosclerotic plaques but also any infected tissues, theirpresence may therefore allow more accurate imaging evaluation of otherpathologies such as, for example, infected bone marrow (Kaim et al.Radiology 2002; 225:808-14), cancer (US Pat Appl 20090004113) and otherdiseases mediated by activated macrophages such as rheumatoid arthritis,ulcerative colitis, Crohn's disease, psoriasis, osteomyelitis, multiplesclerosis, atherosclerosis, pulmonary fibrosis, sarcoidosis, systemicsclerosis, organ transplant rejection (graft-versus-host disease, GVHD)and chronic inflammations (U.S. Pat. No. 7,740,854). Therefore, themacrophages are the most appeling targets for the MRI contrast agents.

Recently, investigators used BSA to deliver Gd to macrophages ex vivoand in vitro (Gustafsson et al. Bioconjug Chem 2006; 17:538-47).However, in vivo data are currently unavailable, and the specificity oftargeting with albumin remains to be seen, because it is a ubiquitoussubstance that is taken up by many tissues and diffuses intointerstitial spaces nonspecifically.

11. Myeloperoxidase as a Target for the MRI Contrast Agents

A possible way to quantify the level of macrophages is to determine theconcentration of myeloperoxidase (MPO), a CD11b-positive cell(neutrophils, macrophages) secreted enzyme, and related components ofthe MPO pathway (Nicholls S. J. & Hazen S. L. Arterioscler Thromb VascBiol 2005; 25:1102-11). Myeloperoxidase (MPO) which emerged as apotential participant in the promotion and/or propagation ofatherosclerosis, is a member of the heme peroxidase superfamily. MPOgenerates numerous reactive oxidants and diffusible radical species(Klebanoff S J. Ann Intern Med 1980; 93:480-9) that are capable of bothinitiating lipid peroxidation (Zhang et al. J Biol Chem 2002;277:46116-22) and promoting an array of post-translational modificationsto target proteins, including halogenation, nitration, and oxidativecross-linking (Heinecke J W. Am J Cardiol 2003; 91:12A-6A).

MPO, the most abundant component of azurophilic granules of leukocytes,is secreted on leukocyte activation, contributing to innate hostdefenses. Found predominantly in neutrophils, monocytes, and somesubtypes of tissue macrophages, MPO amplifies the oxidative potential ofits cosubstrate hydrogen peroxide, forming potent oxidants capable ofchlorinating and nitrating phenolic compounds (Heinecke J W. Am JCardiol 2003; 91:12A-6A; Podrez et al. Free Radic Biol Med 2000;28:1717-25; Gaut et al. J Clin Invest 2002; 109, 1311-9). The hydrogenperoxide substrate may be derived from a number of sources in vivo,including leukocyte NADPH oxidases, xanthine oxidase, uncoupled nitricoxide synthase (NOS), and various Nox isoenzymes. MPO is unique in itsability to generate reactive chlorinating and brominating species suchas hypochlorous acid (HOCl) and hypobromous acids (HOBr), which reactwith electron-rich moieties of a large range of biomolecules (Podrez etal. Free Radic Biol Med 2000; 28:1717-25).

Spurred initially by the recognition that MPO is enriched within humanatheroma (Daugherty et al. 1994; 94:437-44), both MPO and its reactiveoxidants have been implicated as participants in tissue injury during alarge number of inflammatory conditions (Heinecke J W. Am J Cardiol2003; 91:12A-6A; Podrez et al. Free Radic Biol Med 2000; 28:1717-25;Gaut et al. J Clin Invest 2002; 109, 1311-9; Andreadis et al. Free RadicBiol Med 2003; 35:213-25; Heinecke J W. Mechanisms of oxidative damageby myeloperoxidase in atherosclerosis and other inflammatory disorders.J Lab Clin Med 1999; 133:321-5). Multiple lines of evidence suggest thatMPO may play a role in atherogenesis in humans and that proatherogenicbiological consequences may be triggered by oxidative modification oftargets in the artery wall by MPO-generated reactive species.Immunohistochemical and biochemical analyses localize the enzyme and itsoxidation products within human atherosclerotic lesions (Hazell et al.Free Radic Biol Med 2001; 31:1254-62; Hazen S. L. & Heinecke J. W. JClin Invest 1997; 99:2075-81). Lipid oxidation products of plasmalogensgenerated by the MPO-derived oxidant HOCl are both enriched within humanatheroma and possess potent leukocyte chemotactic activity (Thukkani etal. Circulation 2003; 108:3128-33). Incubation of HOCl and low-densitylipoproteins (LDL) results in oxidation of lysine residues inapolipoprotein B-100, the predominant protein of LDL (Hazell L. J. &Stocker R. Biochem J 1993; 290 (Pt 1):165-72). Increased anionic surfacecharge as well as HOCl-induced lipoprotein aggregation, both convert LDLinto a high-uptake form for macrophages, and appear to occur withinhuman atheroma (Hazell et al. J Clin Invest 1996; 97:1535-44).

Under physiological conditions, activated human monocytes also useMPO-generated reactive nitrogen species to render LDL atherogenic,converting it into a high-uptake form for macrophages (Podrez et al. JClin Invest 1999; 103:1547-60) while simultaneously promoting bothapolipoprotein B-100 protein nitration and initiation of LDL lipidperoxidation. The oxidized form of LDL has been demonstrated to beselectively recognized by the scavenger receptor CD36 (Podrez et al. JClin Invest 2000; 105:1095-108), a major participant in fatty streak andatherosclerotic lesion development (Febbraio et al. J Clin Invest 2000;105:1049-56). High density lipoproteins (HDL) isolated fromatherosclerotic lesions contain numerous MPO-derived peptides ofapolipoprotein A-I (apo A-I), the major constituent protein of HDL,including site-specific oxidative modifications by reactive chlorinatingand nitrating species (Zheng et al. J Clin Invest 2004; 114:529-41;Zheng et al. J Biol Chem 2005; 280:38-47). It has been shown thatMPO-catalyzed oxidation of apo A-I preferentially occurs in the arterialwall. Consistent with this finding, immunohistochemical analysis ofhuman atheroma specimens reveals MPO- and HOCl-modified proteinsco-localize with apo A-I in the region of macrophages (Hazell et al. JClin Invest 1996; 97:1535-44).

The correlation between MPO levels and angiographic evidence ofatherosclerotic plaque (Zhang et al. JAMA 2001; 286:2136-42), as well asthe apparent atheroprotective effects of genetic deficiencies of MPO(Asselbergs et al. Am J Med 2004; 116:429-30), are consistent with thehypothesis that MPO participates in the initiation and/or propagation ofcoronary vascular disease (CVD). The ability of systemic MPO levels topredict the likelihood of clinical events suggests that MPO plays a rolein the transition of a mature atherosclerotic plaque to the vulnerablestate.

Activatable paramagnetic MRI contrast agents can be used to directlyimage MPO activity in humans (Nighoghossian et al. Stroke 2005;36:2764-72; Sinusas et al. Circulation: Cardiovascular Imaging 2008;1:244-56). These agents include Gd complexes that, when cleaved by MPO,expose the shielded Gd to water resulting in alterations of T1relaxivity (Louie et al. Nat Biotechnlmol 2000; 18:321-5). A differentapproach involves the derivatization of Gd chelators such asdiethylenetriaminepentaacetic acid-Gd (DTPA-Gd) with 5-hydroxytryptamide[bis-5HT-DTPA(Gd)]. Myeloperoxidase activates the small-moleculesubstrate, which then polymerizes and exhibits increased T1 relaxivity,protein binding, and “trapping” in areas of high myeloperoxidase (MPO)activity, all leading to increased enhancement on T1-weighted MRI(Nahrendorf et al. Circulation 2008; 117:1153-60; Querol et al. Org Lett2005; 7:1719-22). Others have engineered magnetic nanoparticles thatassemble and disassemble, which can be used for detection of enzymaticactivity (Perez et al. Chembiochem 2004; 5:261-4).

12. Macrophage Scavenger Receptor as a Target for Imaging Agents

Another, clinically more promising approach to evaluate plaquemacrophage burden in vivo, is to target MSR, a macrophage-specificcell-surface protein, which is significantly overexpressed onatherosclerotic macrophages and foam cells (Gough et al. ArteriosclerThromb Vasc Biol 1999; 19:461-71; Amirbekian et al. Proc Natl Acad SciUSA 2007; 104:961-6). The MSR is not expressed on normal vessel wallcells (de Winther et al. Arterioscler Thromb Vasc Biol 2000; 20:290-7).The MSR plays an important role in LDL uptake as well as in clearance ofdebris, including necrotic and apoptotic cell fragments (Peiser et al.Curr Opin Immunol 2002; 14:123-8). Such an integral position in thepathogenesis of atherosclerosis makes the scavenger receptor anexcellent target for molecular imaging. There are several reasons forselecting MSR as a target for assessing atherosclerosis. First, MSRplays a key role in the pathogenesis of atherosclerosis and knocking outeither of the MSR receptors results in marked decreases inatherosclerotic plaque size (Suzuki et al. Nature 1997; 386:292-6). Inaddition, MSR is a primary route of lipoprotein uptake, including uptakeof modified lipoproteins such as oxidized LDL (Goldstein et al. ProcNatl Acad Sci USA 1979; 76:333-7). Second, MSR is widely expressed onatheroma-associated macrophages (Gough et al. Arterioscler Thromb VascBiol 1999; 19:461-71), cells that are present through all stages ofatherosclerosis development, from the initiation of plaques through theformation of complex plaques containing foam cells, lipid accumulations,necrotic debris, and thrombus (Hansson G K. N Engl J Med 2005;352:1685-95). A third key reason (for selecting macrophages and MSR) isthat high macrophage content has been specifically associated withplaque vulnerability to rupture and sequelae, including complete vesselobstruction, myocardial infarction, sudden cardiac death, and stroke(Kolodgie et al. N Engl J Med 2003; 349:2316-25). Finally, MSR is ahigh-affinity receptor, in the picomolar to nanomolar range (dependingon the ligand) and is present in great numbers onatherosclerosis-associated macrophages (Gough et al. Arterioscler ThrombVasc Biol 1999; 19:461-71; Krieger M. & Herz J. Annu Rev Biochem 1994;63:601-37).

MSR targeting can be accomplished either by using receptor-specificantibodies that bind to the receptor on the macrophage surface or byemploying MSR-specific ligands that are uptaken by the macrophages viathe MSR-mediated route. Gd-based micelles have been previously developedand used to target macrophages in plaques (Chen et al. Contrast MediaMol Imaging 2008; 3:233-42). Micelles and liposomes have the advantagesof high lipid capacity and high pay-load of GBCAs (Briley-Saebo et al. JMagn Reson Imaging 2007; 26:460-79; Briley-Saebo et al. Circulation2008; 117:3206-15; Mulder et al. Magn Reson Med 2007; 58:1164-70;Lipinski et al. Magn Reson Med 2006; 56:601-10). Antibodies to mouseMSRs, CD204, have been conjugated to this platform as targeting moietiesto form immunomicelles (Mulder et al. Magn Reson Med 2007; 58:1164-70)that provided excellent in vivo enhancement of atherosclerotic plaques,which was thoroughly validated by histology. However, theseimmunomicelles targeting MSRs are rapidly removed from the circulationby the liver because Kupffer cells also express scavenger receptors andplay a prominent role in the uptake of a wide variety of ligands.Therefore targeting the macrophage with relatively inexpensiveMSR-specific ligands appears to be much more attractive than usingmonoclonal antibodies, which are expensive to produce and purify.

13. Delivery Vehicles that can Carry Imaging Agents

In addition to micelles, other nanoparticulate carriers, such asemulsions or liposomes can be potentially used to carry the imagingagent to the site of interest (U.S. Pat. Nos. 7,179,484; 5,676,928;Sanz, J. & Fayad, Z. A. Nature 2008; 451:953-7; US Pat Appl20070243136). However, the size of these liposomes and emulsions is suchthat it exceeds the size required to readily permeate into theextracellular space and hence into a plaque (Sloop et al. J Lipid Res1987; 28:225-37). For example, liposomes typically have a diameter ofabout 100-400 nm and cannot enter a plaque unless the endothelium isdamaged (e.g., Lanza et al. Circulation 2002; 106:2842-7; Li et al.Radiology 2001; 218:670-8). Therefore, delivery of imaging agentsthrough the use of such nanoparticles is practically restricted toeither targets on the endothelium or in lesions in which endothelialintegrity has been breached, for example, after balloon angioplasty(Lanza et al. Circulation 2002; 106, 2842-7).

Reconstituted lipoproteins have previously been used as deliveryvehicles for lipophilic drugs (U.S. Pat. No. 6,306,433). Lipoproteinsare produced mainly by the intestine and liver (or by processing ofintestine or liver-derived lipoproteins) and are the native transportersin the circulation of a variety of lipophilic and hydrophilic compoundsand are classified into four main categories depending on size andcomposition (i.e., in order of decreasing diameter: chylomicrons, verylow density lipoproteins (VLDL), LDL and HDL (Havel et al., TheMetabolic and Molecular Bases of Inherited Disease. New York:McGraw-Hill; 2001:2705-16). With the exception of HDLs, the lipoproteinsalso suffer the same drawbacks as micelles, conventional emulsions andliposomes, in that the entities are too large to serve as good vehiclesfor the delivery of imaging agents.

LDLs are particularly unsuitable for such delivery because, in additionto being larger than the optimal size (on average LDLs are larger than20 nm), the major protein constituent of LDLs is apoB, a very large andhydrophobic protein, which makes it difficult to reconstitute LDL (rLDL)particles. Furthermore, LDL moieties are spontaneously retained inatherosclerotic lesions (Williams K. J. & Tabas I. Arterioscler ThrombVasc Biol 1995; 15:551-61), thereby making it difficult to selectivelydetect specific molecular targets of interest within the plaque. Yetanother factor that makes LDLs unattractive as delivery vehicles is thatLDL is an atherogenic particle, and so it is difficult to justify thepossible risks from administration of rLDL to patients already at highrisk for cardiovascular disease.

Micelles, much like LDLs, also do not serve well as delivery vehicles toenter atherosclerotic plaques, because they are spontaneously retainedfor prolonged periods of time, rendering them unsuitable for theselective detection of specific molecules of interest.

14. Macrophage Targeting Using HDL

Reconstituted HDL (rHDL) and recombinant HDL have been recently used forthe treatment and prevention of acute coronary symptoms, stroke andother disorders (Tardif et al. JAMA 2007; 297:1675-82; Newton R. S. &Krause B. R. Atheroscler Suppl 2002; 3:31-8; Nissen et al. JAMA 2003;290:2292-300; Choudhury et al. Arterioscler Thromb Vase Biol 2004;24:1904-9; U.S. Pat. Nos. 7,435,717; 6,953,840; 7,491,693).

Recently, HDL have been suggested as a specific contrast agent for MRIof atherosclerotic plaques (US Pat Appl 20070243136; Frias et al.Contrast Media Mol Imaging 2007; 2:16-23; Frias et al. J Am Chem Soc2004; 126:16316-7; Cormode et al. Small 2008; 4:1437-44; Chen et al.Contrast Media Mol Imaging 2008; 3:233-42; Frias et al. Nano Lett 2006;6:2220-4). It should be noted that the term “modified lipoproteins” usedby Frias et al. (Frias et al. Contrast Media Mol Imaging 2007; 2:16-23)accurately speaking refers to the lipoproteins labeled (rather thanmodified) with radioisotopes for nuclear imaging, chelates for MRI orother possible contrast agents for computed tomography imagingtechniques. It should be further noted that based on the descriptionprovided in the aforementioned article, protein (apolipoprotein) part ofthe lipoprotein is not labeled or modified. Importantly, the term“modified lipoproteins” is also used in the art to describe lipoproteinsmodified by any means, for example by peroxidation of lipids orHOCl-mediated oxidative modification of proteins by reactivechlorinating and nitrating species. These chemically modifiedlipoproteins represent a high affinity substrate for MSR-mediated uptakeby macrophages whereas “modified lipoproteins” as defined by Frias etal. may not demonstrate this quality (Frias et al. Contrast Media MolImaging 2007; 2:16-23).

In comparison to other Gd-based macrophage targeting platforms (e.g.,LDL and immunomicelles), HDL have several advantages that make themattractive as a specific contrast agent for imaging: they can be readilyreconstituted from their components; they contain an endogenous proteincomponent (apolipoprotein A-I) that does not trigger immunoreactions,and they play a key role in reverse cholesterol transport by removingexcess cellular cholesterol from the macrophages thus demonstrating atherapeutic potential in addition to their imaging function (Forrester,J. S. & Shah, P. K. Am J Cardiol 2006; 98:1542-9; Williams et al. CurrOpin Lipidol 2007; 18:443-50). In plaque imaging, the small size of theHDL particle allows it to enter and accumulate in the plaque. TheHDL-based contrast agen can be obtained as spherical or discoidal formswith similar relaxitivity values. Both forms target atheroscleroticplaques and enhance the MRI signal in a manner dependent on plaquemacrophage content. While not specifically studied, intracellular uptakewould be expected to occur through the MSR for both spherical anddiscoidal forms with no differences in the diffusion of either particleinto the atherosclerotic plaque.

Despite multiple advantages of the HDL nanoparticles as deliveryplatform, the currently suggested HDL compositions for use as imagingagents in MRI, CT, Gamma-scintigraphy, or optical imaging techniques (USPat Appl 20070243136) have low specificity of targeted delivery ofcontrast molecules such as GdBCAs to the plaque and, importantly, lowsubsequent retention within the arterial wall. This results in the lowamount of contrast agent delivered and, therefore, low MRI contrastenhancement which does not significantly reduce the dosage of Gdrequired. In order to increase the delivery and retention of rHDL withinthe arterial wall, antibodies for different plaque components can beincorporated in rHDL as targeting moieties (US Pat Appl 20070243136).However, the suggested imaging agents would share all the disadvantagesof antibodies (unstable, expensive to produce, potentially immunogenic,etc.).

Alternatively, an apo E-derived lipopeptide has been shown to increaseefficacy of the rHDL platform for molecular MR imaging ofatherosclerotic plaques in vivo (Chen et al. Contrast Media Mol Imaging2008; 3:233-42). This synthetic lipopeptide represents a dipalmitoylatedversion of apo E-derived highly positive peptide, which has the aminoacid sequence (LRKLRKRLLR)₂, and is a tandem dimer (141-150)₂ derivedfrom the LDL receptor binding domain of apo E. Despite resulting in animproved in vivo MR imaging signal enhancement in atherosclerotic mice(90% vs. 53% enhancement within the arterial vessel 24 h afteradministration of a 50 micromol Gd/kg dose), incorporation of thisdetergent-like highly positive molecule in the rHDL platform can alsobring the apo E-derived tandem peptide-associated disadvantages to theplatform. For example, this tandem peptide and its dipalmitoylatedversion are known to mediate uptake of liposomes or micelles intoendothelial cells of brain microvessels (Keller et al. Angew Chem Int EdEngl 2005; 44:5252-5; Sauer et al. Biochemistry 2005; 44:2021-9; Saueret al. Biochim Biophys Acta 2006; 1758:552-61). In addition, this tandempeptide can exert neurotoxic effects (Wang X. S. & Gruenstein E. J CellPhysiol 1997; 173:73-83).

Recently, Gd-containing HDL obtained by incubation of native human HDL(commercially available HDL preparations purified from human plasma;Calbiochem, San Diego, Calif.) withGd-DTPA-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (Gd-DTPA-DMPE)and Gd/6-amino-6-methylperhydro-1,4-diazepinetetraacetic acid with a17-carbon long aliphatic chain (Gd-AAZTA-C17) have been suggested ashigh-relaxitivity MRI contrast agents (Briley-Saebo et al. J Phys Chem B2009; 113:6283-9). However, incubation of native HDL with Gd-DTPA-DMPEresulted in the uncontrolled particle fusion due to detergentperturbations whereas the composition and integrity of HDL-Gd-AAZTA-C17adducts was not characterized. In contrast to rHDL platform, both nativeHDL-based agents lack the control and reproducibility between batchesbecause native HDL is a heterogeneous lipoprotein class with differentsubspecies that vary in apolipoprotein and lipid composition, in sizeand charge, and in physiological functions (Castro G. R. & Fielding C.J. Biochemistry 1988; 27:25-9; Miida et al. Biochemistry 1992;31:11112-7; Mowri et al. J Lipid Res 1992; 33:1269-79; von Eckardsteinet al. Curr Opin Lipidol 1994; 5:404-16). For this reason, the size,shape, protein and lipid composition, structure, properties andphysiological function of native HDL purified from human plasma usingultracentrifugation vary significantly depending on donors, isolationprocedure variations and storage conditions and therefore cannot be wellcontrolled.

15. An Unmet Need

Hence, there is a need for a targeted delivery vehicle that can freelyenter an atherosclerotic plaque or other sites of interest such as tumorsites and that provides sufficient quantities of an imaging agent tomeet the needs of MRI or MR spectroscopy or other imaging techniquessuch as CT, gamma-scintigraphy, and optical, positron emissiontomography (PET), and combined imaging techniques. This agent shouldpossess a high affinity for macrophages and their components in order tosignificantly reduce the contrast agent dosage required and thus limitconcerns related to systemic toxicity, which is especially important forGd-based contrast agents.

SUMMARY OF THE INVENTION

The present invention provides various nanoparticles that containchemically and/or enzymatically modified apolipoproteins and are usedfor the delivery of an imaging contrast agent. The compositions of thepresent invention include, but are not limited to, a syntheticnanoparticle, the synthetic nanoparticle comprising at least onechemically and/or enzymatically modified apolipoprotein (apo) A-I and/orA-II, at least one amphipathic lipid, and at least one metallic ornon-metallic contrast agent linked through a chelator to a component ofthe nanoparticle or encapsulated into the nanoparticle, the one metallicor non-metallic contrast agent being present in an amount of between 5%to about 50% (w/w) of the nanoparticle, and the synthetic nanoparticlehaving a diameter of from about 5 nm to about 50 nm. It is contemplatedthat greater amounts of contrast agent e.g., up to 100% (w/w) of thecomponent of the nanoparticle to which the contrast agent is bonded maybe present. Furthermore, while the size of the nanoparticles ispreferably between 5 nm and 25 nm, the diameter may be up to 150 nm. Thenanoparticles may be spherical, discoidal or a distorted disc shape,e.g., ellipsoidal.

BRIEF DESCRIPTION OF THE FIGURES

The following figures form part of the present specification and areincluded to further illustrate aspects of the present invention. Theinvention may be better understood by reference to the figures incombination with the detailed description of the specific embodimentspresented herein.

FIG. 1 presents a schematic representation of one embodiment of adiscoidal imaging agent of the present invention. Chelated contrastagent is shown for illustrative purposes. The contrast agent may or maynot be the chelated one.

FIG. 2A presents a schematic representation of one embodiment of aspherical imaging agent of the present invention with surface-boundcontrast agents. Chelated contrast agent is shown for illustrativepurposes. The contrast agent may or may not be the chelated one.

FIG. 2B presents a schematic representation of one embodiment of aspherical imaging agent of the present invention with encapsulatedcontrast agents. Chelated contrast agent is shown for illustrativepurposes. The contrast agent may or may not be the chelated one.

FIG. 3A illustrates a hypothesized molecular mechanism of action of animaging agent of the present invention as applied to cardiovascularimaging. While not being bound to any particular theory, it is believedthat chemical and/or enzymatic modification of protein constituents ofhigh density lipoprotein (HDL) particles of the present invention leadsto the recognition of these particles by the macrophage scavengerreceptors and results in an irreversible binding to and consequentuptake by macrophages of such HDL particles. It is further believed thataccumulation of these particles in the macrophages is accompanied byaccumulation of contrast agents (CAs) covalently or non-covalently boundto the particles. In contrast, HDL particles that contain onlyunmodified apolipoprotein molecules are not recognized by macrophagesand return to the circulation. Chelated contrast agent is shown forillustrative purposes. The contrast agent may or may not be the chelatedone.

FIG. 3B illustrates a hypothesized molecular mechanism of action of animaging agent of the present invention as applied to cancer imaging.While not being bound to any particular theory, it is believed thatchemical and/or enzymatic modification of protein constituents of highdensity lipoprotein (HDL) particles of the present invention leads tothe recognition of these particles by the macrophage scavenger receptorsand results in an irreversible binding to and consequent uptake bytumor-associated macrophages of such HDL particles. It is furtherbelieved that accumulation of these particles in the macrophages isaccompanied by accumulation of contrast agents (CAs) covalently ornon-covalently bound to the particles. In contrast, HDL particles thatcontain only unmodified apolipoprotein molecules are not recognized bymacrophages and return to the circulation. Chelated contrast agent isshown for illustrative purposes. The contrast agent may or may not bethe chelated one.

FIG. 4 contains examples of naturally occurring chemical and enzymaticmodifications of amino acid residues of proteins including, but notlimited to, apolipoproteins of the present invention and fragmentsthereof. Abbreviation used: MPO—myeloperoxidase.

FIG. 5A presents a schematic representation of one possibleapolipoprotein (apo) of the present invention—apo A-I. Three methionine(Met) residues at positions 86, 112, and 148 and one tyrosine (Tyr)residue at position 192 that most often undergo chemical and/orenzymatic modifications are shown.

FIG. 5B presents a schematic representation of several modifications ofapolipoprotein (apo) A-I naturally occurring in vivo and artificiallyproduced in vitro. While not being bound to any particular theory, it isbelieved that Met residues at positions 112 and 148 are the major sitesof sulfoxidation by hydrogen peroxide alone or in combination withmyeloperoxidase in vivo, whereas sulfoxides of all three Met residues ofapo A-I can be produced in different combinations in vitro, depending onthe oxidant used. It is also believed that Tyr residue at the position192 is the major of nitration and chlorination by myeloperoxidase invivo and in vitro.

FIG. 6A presents the exemplary data showing isolation of pooledapolipoproteins A-I- and A-II-containing fractions from delipidated1.063-1.210 g/ml human high density lipoproteins (HDL) using a ToyopearlHW-55F chromatography. Elution buffer: 10 mM Tris-HCl, 8 M urea, pH 8.6.

FIG. 6B presents the exemplary data showing purification ofapolipoproteins A-I and A-II from pooled apolipoproteins A-I- andA-II-containing fractions using a DEAE-Toyopearl 650M chromatography.Starting buffer, 10 mM Tris-HCl, 0.02 M sodium chloride (NaCl), 8 Murea, pH 8; linear gradient of NaCl from 0.02 to 0.15 M in the samebuffer, total gradient volume 1000 ml; flow-rate, 60 ml/h.

FIG. 7 presents the exemplary data demonstrating purity and homogeneityof apolipoproteins (apo) purified from human serum. SDS-PAGE in 15%polyacrylamide gel of purified apo A-1 and apo A-II. Lanes:1—low-molecular-mass standards from Pharmacia (molecular masses of thestandards are those provided by Pharmacia (×10⁻³); 2, 3, 4-10 ug, 10 ug(with 2% of 2-mercaptoethanol) and 30 ug of purified apo A-I,respectively; 5, 6, 7-10 ug, 10 ug (with 2% of 2-mercapioethanol) and 30ug of purified apo A-II, respectively. Apo A-I and apo A-II areidentified by comparison with known standards.

FIG. 8 presents the exemplary data showing the electrophoretic assaysuitable for the quantitation of apolipoprotein (apo) A-I in fresh,frozen and lyophilized serum pools. Non-reducing SDS-PAGE of 0.5, 1.0and 2.0 ug of apo A-I (lanes 1-3) along with fresh (lanes 4-7), frozen(lanes 8-13) and lyophilized (lanes 14-17) different serum samples.

FIG. 9 presents the exemplary data showing calibration curves preparedwith three samples of the pooled serum: fresh (open circles), frozen(filled circles), and lyophilized (triangles) samples. The data forpurified apolipoprotein A-I is shown using open squares with dots. Allsamples were diluted as follows: 1:29 (1), 1:14 (2), 1:9 (3), 1:6.5 (4)and 1:2.75 (5).

FIG. 10A presents the exemplary data showing an analyticalreversed-phase HPLC profile of the initial apo A-I isolated and purifiedfrom human serum. The retention times (in minutes) are shown above eachpeak.

FIG. 10B presents the exemplary data showing an analyticalreversed-phase HPLC profile of apo A-I_(unox) isolated from the initialapo A-I by preparative reversed-phase HPLC. The retention times (inminutes) are shown above each peak.

FIG. 10C presents the exemplary data showing an analyticalreversed-phase HPLC profile of apo A-I_(ox) obtained by treatment of apoA-I_(unox) with hydrogen peroxide for 15 min and subsequent isolation bypreparative reversed-phase HPLC. The retention times (in minutes) areshown above each peak.

FIG. 10D presents the exemplary data showing an analyticalreversed-phase HPLC profile of apo A-I_(ox) after incubation withpeptide methionine sulfoxide reductase for 60 min. The retention times(in minutes) are shown above each peak.

FIG. 10E presents the exemplary data showing an analyticalreversed-phase HPLC profile of apo A-I_(red) isolated by preparativereversed-phase HPLC. The retention times (in minutes) is shown above thepeak.

FIG. 11A presents the exemplary data showing 12.5% SDS-PAGE analysis ofpurified unoxidized, oxidized and reduced apo A-I (lanes 1, 2, and 3,respectively). The positions and molecular masses of protein standardsare indicated on the left of the gels.

FIG. 11B presents the exemplary data showing 15% SDS-PAGE analysis ofunoxidized, oxidized and reduced apo A-I digested with CNBr (lanes 1, 2,and 3, respectively). The positions and molecular masses of proteinstandards are indicated on the left of the gels.

FIG. 11C presents the exemplary data showing 15% SDS-PAGE analysis ofunoxidized, oxidized and reduced apo A-I digested with chymotrypsin(lanes 1, 2, and 3, respectively). The positions and molecular masses ofprotein standards are indicated on the left of the gels.

FIG. 12A presents the exemplary data showing far-UV circular dichroismspectra of 7.2 uM unoxidized apo A-I with (dotted line) and without(solid line) 2.3 mM 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC);and in the presence of 4 M urea (dashed line) in 10 mM ammoniumbicarbonate, 0.005% sodium azide, pH 7.8; in a 1 mm path-length cell at25° C., with 1 nm bandwidth and 1.0 s averaging per point.

FIG. 12B presents the exemplary data showing far-UV circular dichroismspectra of 7.2 uM oxidized apo A-I with (dotted line) and without (solidline) 2.3 mM 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC); and inthe presence of 4 M urea (dashed line) in 10 mM ammonium bicarbonate,0.005% sodium azide, pH 7.8; in a 1 mm path-length cell at 25° C., with1 nm bandwidth and 1.0 s averaging per point.

FIG. 12C presents the exemplary data showing far-UV circular dichroismspectra of 7.2 uM reduced apo A-I with (dotted line) and without (solidline) 2.3 mM 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC); and inthe presence of 4 M urea (dashed line) in 10 mM ammonium bicarbonate,0.005% sodium azide, pH 7.8; in a 1 mm path-length cell at 25° C., with1 nm bandwidth and 1.0 s averaging per point.

FIG. 12D presents the exemplary data showing temperature-inducedunfolding spectra collected in a temperature range of 25-95° C. at 222nm on a solution of 7.2 uM unoxidized, oxidized, and reduced apo A-I in10 mM ammonium bicarbonate, 0.005% sodium azide, pH 7.8; in a 1 mmpath-length cell; with 1 nm bandwidth, 1° C. temperature increment, and5.0 s averaging per point.

FIG. 13 presents the exemplary data showing far-UV circular dichroismspectra of apo A-I on different rHDL particles. The spectra oflipid-associated apo A-I in rHDL-1 (open circles), rHDL-2 (opensquares), rHDL-3 (open diamonds) and rHDL-4 (crossings) were recordedfrom 190 to 260 nm at 25° C. using a 1 mm path-length quartz cuvette onAVIV 62A DS spectropolarimeter. The samples were analyzed at a proteinconcentration of 1.8 uM (0.05 mg of protein/ml) rHDL particles in TBS,pH 7.4, and spectra of at least six scans were signal averaged andbaseline corrected by subtracting an averaged buffer spectrum.

FIG. 14 presents the exemplary data showing denaturation by GuanidineHydrochloride (GdnHCl) of lipid-free apo A-I and rHDL complexes.Aliquots of unoxidized (open circles) and oxidized (open squares)lipid-free apo A-I proteins or prepared rHDL-1 (filled circles), rHDL-2(filled squares), rHDL-3 (filled diamonds) and rHDL-4 (filledupside-down triangles) complexes were incubated at 4° C. with 0-6 MGdnHCl in 10 mM TBS, pH 7.4 for 72 h. The fluorescence intensities at353 and 333 nm were measured at 25° C. using a 4×4 mm2 cuvette on aFluoro-Max-2 spectrofluorimeter with sample protein concentrationsbetween 0.05 and 0.1 mg of protein/ml. The emission spectra were takenby exciting at 285 nm with a resolution of 2 nm and by measuring theemission with a resolution of 4 nm. The ratio of fluorescence intensityat 353 nm to that at 333 nm is plotted against the GdnHCl molarconcentration.

FIG. 15 presents the exemplary data showing the free energy ofdenaturation of lipid-free apo A-I and rHDL complexes as a function ofthe ionic activity of GdnHCl. The free energy values of denaturation(calculated from change in the ratio of fluorescence intensity at 353 nmto that at 333 nm) are fitted using linear regression equations andplotted against RT ln(1+Ka) for unoxidized (open circles) and oxidized(open squares) lipid-free apo A-I proteins or prepared rHDL-1 (filledcircles), rHDL-2 (filled squares), rHDL-3 (filled diamonds) and rHDL-4(filled upside-down triangles) complexes. The standard free energy ofdenaturation (ΔG_(D) ⁰) and the number of the bound GdnHCl moles (Δn)were computed from the intercepts on the vertical axis and the slopes ofthe regression lines, respectively, as described by Sparks et al.(Sparks et al. J Biol Chem 1992; 267:25839-47).

FIG. 16 presents the exemplary data showing temperature-inducedunfolding of rHDL complexes. The circular dichroic data were collectedat 222 nm every 2-5° C. from 25 to 95° C. on solutions of 1.8 uM (0.05mg of protein/ml) prepared rHDL-1 (filled circles), rHDL-2 (filledsquares), rHDL-3 (filled diamonds) and rHDL-4 (filled upside-downtriangles) complexes in TBS, pH 7.4, with a 1 mm path-length quartzcuvette on AVIV 62A DS spectropolarimeter.

FIG. 17 presents the exemplary data showing SDS-PAGE analysis of thetrypsin digestion products of rHDL complexes. The rHDL complexes weretreated by trypsin at room temperature for 2 h. Lane 1—rHDL-1 containingonly apo A-I_(unox). Lane 2—rHDL-2 containing only apo A-I_(ox). Lane3—rHDL-3 containing apo A-I_(unox) and apo A-I_(ox) with a molar ratioof 1:1. Lane 4—rHDL-4 containing apo A-I_(unox), apo A-I_(ox) and apoA-II_(unox) with a molar ratio of 3:3:1. The positions and molecularweights of the protein standards are indicated on the right of the gel.The 14 kDa fragment is identified with an asterisk mark.

FIG. 18 presents the exemplary data showing1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) kinetic binding withunoxidized, oxidized, and reduced apo A-I proteins. DMPC solubilizedabove its transition temperature (>24° C.) in TBS, pH 8.0, was dilutedby the same buffer to 0.5 mg/ml and preincubated for 10 min at 24° C.Then unoxidized (filled circles), oxidized (open circles), and reduced(filled triangles) apo A-I proteins dissolved in the same buffer wereadded (final DMPC/protein molar ratio, 50:1), and the reaction wasfollowed at 24° C. for 30 min at 325 nm.

FIG. 19A presents the exemplary data showing an analyticalreversed-phase HPLC profile of apo A-I in rHDL particles containing onlyapo A-I_(unox) (rHDL-1). The retention time (in minutes) is shown abovethe peak.

FIG. 19B presents the exemplary data showing an analyticalreversed-phase HPLC profile of apo A-I in rHDL particles containing onlyapo A-I_(ox) (rHDL-2) before (solid line) and after (dotted line) thetreatment by the peptide methionine sulfoxide reductase (PMSR) enzyme inthe presence of dihydrolipoic acid (DHLA). The retention times (inminutes) are shown above each peak.

FIG. 19C presents the exemplary data showing an analyticalreversed-phase HPLC profile of apo A-I in rHDL particles containing apoA-I_(unox) and apo A-I_(ox) with a molar ratio of 1:1 (rHDL-3) before(solid line) and after (dotted line) the treatment by the peptidemethionine sulfoxide reductase (PMSR) enzyme in the presence ofdihydrolipoic acid (DHLA). The retention times (in minutes) are shownabove each peak.

FIG. 19D presents the exemplary data showing an analyticalreversed-phase HPLC profile of apo A-I in rHDL particles containing apoA-I_(unox), apo A-I_(ox), and apo A-II_(unox) with a molar ratio of3:3:1 (rHDL-4) in TBS, pH 7.4. The retention times (in minutes) areshown above each peak.

FIG. 19E presents the exemplary data showing an analyticalreversed-phase HPLC profile of apo A-I in rHDL particles containing apoA-I_(unox), apo A-I_(ox), and apo A-II_(unox) with a molar ratio of3:3:1 (rHDL-4) in the enzyme reaction buffer containing DHLA in theabsence of the enzyme (solid line) and after PMSR/DHLA treatment (dottedline). As DHLA reduces the disulfide bond in native apo A-II dimer, itdissociates into identical 77-residue monomers. The retention time ofthis apo A-II monomer is similar to that for apo A-I_(unox). Theretention times (in minutes) are shown above each peak.

FIG. 20 presents the exemplary data showing observed values of relativecholesterol efflux from cholesterol-loaded human skin fibroblastspromoted by HDL containing unmodified (unoxidized) apo A-I (empty bars)and oxidized (apo A-I₊₃₂, or apo A-I_(Met112SO,Met148SO)) apo A-I (solidbars). Concentrations of HDL were as follows: A—10 mg of apolipoproteinA-1 per liter of medium; B—25 mg of apolipoprotein A-I per liter ofmedium.

DETAILED DESCRIPTION OF THE INVENTION Definitions

It is understood by a person of ordinary skill in the art that the terms“APOA1_HUMAN”, “Apolipoprotein A-I”, “Apolipoprotein A-1”, “APOA1”,“ApoA-I”, “Apo-A1”, “ApoA-1”, “apo-A1”, “apoA-1” and “Apo-A1” refer tothe naturally occurring human protein listed in the UniProtKnowledgebase (UniProtKB, www.uniprot.org) under the name “APOA1_HUMAN”.The protein amino acid sequence can be found under the entry UniProtKB/Swiss-Prot P02647 (www.uniprot.org/uniprot/P02647, last modified onJul. 28, 2009, version 137). It is further understood by the person ofordinary skill in the art that the terms “APOA2_HUMAN”, “ApolipoproteinA-II”, Apolipoprotein A-2”, “APOA2”, “ApoA-II”, “Apo-AII”, “ApoA-2”,“apo-A2”, “apoA-2” and “Apo-A2” refer to the naturally occurring humanprotein listed in the UniProt Knowledgebase (UniProtKB, www.uniprot.org)under the name “APOA2_HUMAN”. The protein amino acid sequence can befound under the entry UniProt KB/Swiss-Prot P02652(http://www.uniprot.org/uniprot/P02652, last modified on Jul. 28, 2009,version 121).

As understood by those of ordinary skill in the art, the amino acidcomposition of the human apo A-I can be determined by reviewing theUniProtKB entry which describes the naturally occurring unmodifiedprotein that consists of 267 amino acids and has a molecular weight of30,778 Dalton. As further understood by those of ordinary skill in theart, the amino acid composition of the human apo A-II can be determinedby reviewing the UniProtKB entry which describes the naturally occurringunmodified protein that consists of 100 amino acids and has a molecularweight of 11,175 Dalton. As employed herein, the term “apo A-II”describes apo A-II either in homodimeric or monomeric form.

It is understood by the ordinary skill in the art that when apo A-I ismodified such that its molecular weight changes, the resultant modifiedprotein is referred to as either apoA-1 (+X) wherein “X” is the increasein protein's molecular weight in Daltons or as apoA-I (−Y) wherein “Y”is the decrease in protein's molecular weight in Daltons (Garner et al.J Biol Chem 1998; 273:6080-7; Pankhurst et al. J Lipid Res 2003;44:349-55). It is further understood by the ordinary skill in the artthat when apo A-II is modified such that its molecular weight changes,the resultant modified protein is referred to as either apoA-II (+X)wherein “X” is the increase in protein's molecular weight in Daltons oras apoA-II (−Y) wherein “Y” is the decrease in protein's molecularweight in Daltons (Garner et al. J Biol Chem 1998; 273:6080-7; Pankhurstet al. J Lipid Res 2003; 44:349-55).

As employed herein and understood by the ordinary skill in the art theterm “recombinant protein” describes the protein obtained from bacterialor other sources using the recombinant DNA technology (Nissen et al.JAMA 2003; 290:2292-300). Furthermore, a suffix or a prefix indicatingthe species from which the protein is derived is added to the protein'sname when a non-human protein such as non-human apo A-I or apo A-II isdescribed (U.S. Pat. No. 6,953,840;http://www.uniprot.org/uniprot/Q00623;http://www.uniprot.org/uniprot/P09813). In special cases a suffix or aprefix may also indicate a well-known apoA-1 variant, e.g. apo A-IMilano (U.S. Pat. No. 7,435,717; Nissen et al. JAMA 2003; 290:2292-300).As used herein, the term “aptamer” or “specifically bindingoligonucleotide” refers to an oligonucleotide that is capable of forminga complex with an intended target substance. The complexation istarget-specific in the sense that other materials which may accompanythe target do not complex to the aptamer. It is recognized thatcomplexation and affinity are a matter of degree; however, in thiscontext, “target-specific” means that the aptamer binds to target with amuch higher degree of affinity than it binds to contaminating materials.The term “peptidomimetic” as used herein refers to a peptide-likemolecule containing non-hydrolyzable chemical moieties in place of oneor more hydrolyzable moieties existing in naturally occurring peptides.Thus, regions of a peptide which are hydrolyzable, such as carboxylmoieties, are replaced by non-hydrolyzable moieties, such as methylenemoieties, in a peptidomimetic.

In the present invention, the term “modified protein” is used todescribe chemically or enzymatically or chemically and enzymaticallymodified oligopeptides, oligopseudopeptides, polypeptides,pseudopolypeptides, and native proteins (synthetic or otherwisederived), regardless of the nature of the chemical and/or enzymaticmodification. The term “pseudopeptide” refers to a peptide where one ormore peptide bonds are replaced by non-amido bonds such as ester or oneor more amino acids are replaced by amino acid analogs. The term“peptides” refers not only to those comprised of all natural aminoacids, but also to those which contain unnatural amino acids or othernon-coded structural units. The terms “peptides”, when used alone,include pseudopeptides. It is worth mentioning that “modified proteins”have utility in many biomedical applications because of their increasedstability against in vivo degradation, superior pharmacokinetics, andaltered immunogenecity compared to their native counterparts.

The term “modified protein,” as employed herein, also includes oxidizedproteins. The term “oxidized protein” refers to a protein in which atleast one amino acid residue is oxidized. The term “oxidized proteinfragment” refers to a protein fragment in which at least one amino acidresidue is oxidized. The term “oxidation status” refers to a metric ofthe extent to which specific amino acid residues are replaced bycorresponding oxidized amino acid residues in a protein or a proteinfragment. The term “extent of oxidation” refers to the degree to whichpotentially oxidizable amino acids in a protein or fragment haveundergone oxidation. For example, if the protein fragment contains asingle tyrosine residue which is potentially oxidized to3-chlorotyrosine, then an increase in mass of about 34 Dalton (i.e., theapproximate difference in mass between chlorine and hydrogen) indicatesoxidation of tyrosine to 3-chlorotyrosine (Pitt, A. R. and Spickett, C.M. 2008; 36:1077-82; Shao et al. J Biol Chem 2006; 281:9001-4; Shao etal. J Biol Chem 2005; 280:5983-93). Similarly, if the protein fragmentcontains a single methionine residue which is potentially oxidized tomethionine sulfoxide, then an increase in mass of 16 Dalton (i.e., thedifference in mass between methionine and methionine containing oneextra oxygen) indicates oxidation of methionine to methionine sulfoxides(Garner et al. J Biol Chem 1998; 273:6080-7).

The oxidation status can be measured by metrics known to the arts ofprotein and peptide chemistry (US Pat Appl 20080020400; US Pat Appl20050239136) including, without limitation, assay of the number ofoxidized residues, mass spectral peak intensity, mass spectralintegrated area, and the like. In some embodiments of any of the aspectsprovided herein, oxidation status is reported as a percentage, wherein0% refers to no oxidation and 100% refers to complete oxidation ofpotentially oxidizable amino acid residues within apo A-I or apo A-II orfragments thereof. The term “potentially subject to oxidation,”“potentially oxidizable amino acid residues”, and the like refer to anamino acid which can undergo oxidation, for example by nitration orchlorination.

In the context of the present invention, the term “oxidation fraction”refers to the term “oxidation status” as defined herein expressed as afraction in the range 0-1, e.g., 0.0, 0.1, 0.2, 0.3, and the like up to1.0. It is understood that the number of significant digits in anoxidation status, oxidation fraction, or other experimental resultherein is a function of the sensitivity of the instruments andexperimental protocols and can assume values of 1, 2, 3, 4, or even moresignificant digits. Methods for the calculation of significant digitsare well known in the art. The phrase “determining the oxidationfraction” refers to determining the oxidation status, as defined herein,and expressing the result as a fraction of amino acids of the protein orprotein fragment which can be replaced by corresponding oxidized aminoacid residues. The term “total amount of protein in a sample” and liketerms refer to the total amount of protein irrespective of the oxidationstate of the constituent amino acids thereof. In some embodiments ofthis aspect, the total amount of apoA-I or A-II is determined by assay,for example without limitation, a standard immunoassay well known in theart. Examples of reagents readily available for immunometricdetermination of apo A-I and A-II include antiserum to apo A-I and A-II,respectively (Dade Behring, Deerfield, Ill.). In some embodiments ofthis aspect, the total amount of apoA-I or A-II is multiplied by theoxidation fraction to provide a quantitation of the amount of oxidizedapoA-I or A-II in a biological sample. For example without limitation,if the oxidation fraction were 0.5 and the total concentration of apoA-I or A-II in the biological sample were 1.0 mg/mL, the amount ofoxidized apoA-I or A-II in the biological sample would be reported as0.5 mg/mL (i.e., 0.5.times.1.0 mg/mL).

The term “encapsulation” as used herein refers to the enclosure of amolecule, such as a contrast agent or therapeutics, inside thenanoparticle. Such encapsulation may be generated, according to anembodiment, by synthesis of nanoparticles in the presence of a liquidsolution containing a contrast agent or therapeutics. The term“incorporation” as used herein refers to imbibing or adsorbing thecontrast agent or therapeutics onto the nanoparticle. A “site ofinterest” on a target as used herein is a site to which modifiedproteins and protein fragments of the present invention bind. The term“target site”, as used herein, refers to sites/tissue areas of interest.As used in this invention, the terms “target cells” or “target tissues”refer to those cells or tissues, respectively that are intended to bevisualized in imaging techniques such as computed tomography (CT),gamma-scintigraphy, positron emission tomography (PET), single photonemission computed tomography (SPECT), magnetic resonance imaging (MRI),and combined imaging techniques, using the compositions of the presentinvention delivered in accord with the invention. Target cells or targettissues take up or link with the modified proteins or protein fragmentsof the invention. Target cells are cells in target tissue, and thetarget tissue includes, but is not limited to, atherosclerotic plaques,vascular endothelial tissue, abnormal vascular walls of tumors, solidtumors, and other tissues or cells related to cardiovascular,inflammatory, and autoimmune disease. Further, target cells includevirus-containing cells, and parasite-containing cells. Also includedamong target cells are cells undergoing substantially more rapiddivision as compared to non-target cells. The term “target cells” alsoincludes, but is not limited to, microorganisms such as bacteria,viruses, fungi, parasites, and infectious agents. Thus, the term “targetcell” is not limited to living cells but also includes infectiousorganic particles such as viruses. “Target compositions” or “targetbiological components” include, but are not be limited to: toxins,peptides, polymers, and other compounds that may be selectively andspecifically identified as an organic target that is intended to bevisualized in imaging techniques using the compositions of the presentinvention. The term “macrophage-related diseases” include diseasesassociated with macrophages such as atherosclerosis and other diseaseassociated with abnormal cholesterol metabolism. Examples ofmacrophage-related diseases, include, but are not limited to, heartdisease, peripheral artery disease, and stroke (e.g., ischemic stroke,hemorrhagic stroke). Other examples include the cancers: sarcoma,lymphoma, leukemia, carcinoma and melanoma, and other activatedmacrophage-related disorders including autoimmune diseases (e.g.,rheumatoid arthritis, Sjogrens, scleroderma, systemic lupuserythematosus, non-specific vasculitis, Kawasaki's disease, psoriasis,Type I diabetes, pemphigus vulgaris), granulomatous diseases (e.g.,tuberculosis, sarcoidosis, lymphomatoid granulomatosis, Wegener'sgranulomatosus), inflammatory diseases (e.g., inflammatory lung diseasessuch as interstitial pneumonitis and asthma, inflammatory bowel diseasesuch as Crohn's disease, and inflammatory arthritis), and transplant(e.g., heart/lung transplants) rejection reactions. The term “plaque”includes, for example, an atherosclerotic plaque.

DETAILED DESCRIPTION

Because of the leading position of cardiovascular disease as a cause ofmortality in industrialized societies, applications in this area arethus highlighted. However, it should be noted that the techniques andcompositions listed and described below are applicable to a broad rangeof disease states such, for example, as cancer and multiple sclerosis.Other features and advantages of the invention will become apparent fromthe following detailed description. It should be understood, however,that the detailed description and the specific examples, whileindicating preferred embodiments of the invention, are given by way ofillustration only, because various changes and modifications within thespirit and scope of the invention will become apparent to those skilledin the art from this detailed description.

Magnetic resonance imaging (MRI) has been effectively used as anon-invasive method for cancer imaging and for the quantification ofatherosclerosis to document its progression and regression ofatherosclerosis in vivo (Skinner et al. Nat Med 1995; 1, 69-73; Corti etal. J Am Coll Cardiol 2002; 39:1366-73; Helft et al. Circulation 2002;105:993-8; Toussaint et al. Circulation 1996; 94:932-8; Cai et al.Circulation 2002; 106:1368-73; Weissleder, R. Nat Rev Cancer 2002;2:11-8). However, significant progress is still needed in spatial andtemporal resolution of plaque characteristics and in the molecularimaging of plaque components. Such progress will be greatly facilitatedby the availability of novel imaging compositions that are small enoughto freely enter an atherosclerotic plaque in sufficient quantities toprovide an enhanced MR image. These particles should preferably possessthe following properties be a) small enough to readily penetrate intothe interstitial fluid, b) able to carry large amounts of a contrastagent, c) non-toxic including non-atherogenic, d) poorly retained indiseased tissue without the addition of a targeting agent, e) able tocarry large amounts of a targeting agent, and f) easy to manufacture andstore.

Chemical or enzymatic modification of fully assembled HDL particles(without Gd) has been shown to enhance their absorption by themacrophages (Bergt et al. Biochem J 2000; 346 Pt 2:345-54; Pankhurst etal. J Lipid Res 2003; 44:349-55; Panzenboeck et al. J Biol Chem 1997;272:29711-20; Suc et al. J Cell Sci 2003; 116:89-99). However, publisheddata demonstrate that in the modified HDL particle described in(Panzenboeck et al. J Biol Chem 1997; 272:29711-20) both, the proteinand the lipid portion of the particle have undergone the chemicalmodification. The prior art (US Pat Appl 20070243136) neither suggestsnor teaches one of ordinary skill in the art to investigate theperformance of HDL particles in which only the apolipoprotein portionhas been chemically altered.

Surprisingly advantageous compositions are demonstrated by the presentinvention which meet the requirements mentioned above. Compositions ofthe invention are rHDL, protein constituents of which, apolipoproteinsA-I and/or A-II or fragments thereof are modified. Certain controlledchemical or enzymatic modification of apolipoproteins (apo) A-I or A-IIor fragments thereof converts these apolipoproteins to substrates formacrophage scavenger receptors and results in the improvement ofassociation of the Gd-(HDL/modified apolipoprotein)-particle withmacrophages and/or absorption (uptake) of the Gd-(HDL/modifiedapolipoprotein)-particle by macrophages when compared to that of theGd-(HDL/apolipoprotein)-particle constructed with non-modified naturallyoccurring apo A-I, apo A-II or fragments thereof. These advantageouscompositions are demonstrated by the present invention to solve numerousproblems which otherwise are associated with high dosages of Gd andother contrast agents required and the lack of control andreproducibility of formulations, especially in large-scale production.

In preferred embodiments, the modified apolipoprotein is selected from amodified apo A-I or a fragment thereof and a modified apo A-II or afragment thereof. In preferred embodiments, the modified apolipoproteinis any combination of a modified apo A-I and a modified A-II andfragments thereof. In preferred embodiments, a modified apo A-I is anoxidized apoA-I or an oxidized apoA-I fragment that comprises one ormore of the following amino acid residues: 3-chlorotyrosine,3-nitrotyrosine, 3,5-dibromotyrosine, dityrosine,trihydroxyphenylalanine, dihydroxyphenylalanine, methionine sulphoxide,and tyrosine peroxide. In still other preferred embodiments, a modifiedapo A-II is an oxidized apoA-II or an oxidized apoA-II fragment thatcomprises one or more of the following amino acid residues:3-chlorotyrosine, 3-nitrotyrosine, 3,5-dibromotyrosine, dityrosine,trihydroxyphenylalanine, dihydroxyphenylalanine, methionine sulphoxide,and tyrosine peroxide. In particularly preferred embodiments, a modifiedapo A-I is an oxidized apo A-I or an oxidized apoA-I fragment thatcomprises methionine sulfoxide at any one of positions 86, 112, 148, orany combination of said positions. In still particularly preferredembodiments, a modified apo A-II is an oxidized apo A-II or an oxidizedapoA-II fragment that comprises methionine sulfoxide at position 26. Instill preferred embodiments apo A-I_(unox) is unoxidized apo A-Icontained in initial serum apo A-I. In other preferred embodiments apoA-II_(unox) is unoxidized apo A-II contained in initial serum apo A-II.In other preferred embodiments, apo A-I_(ox) is oxidized apo A-I (withsulfoxidized methionines at positions 112 and 148) contained in serumapo A-I or obtained from unoxidized apo A-I using hydrogen peroxide. Instill other preferred embodiments; apo A-I_(red) is reduced apo A-Iobtained by reduction of oxidized apo A-I (with sulfoxidized methioninesat positions 112 and 148) using peptide methionine sulfoxide reductase(PMSR). In preferred embodiments, rHDL-1 are reconstituted HDL particlescontaining only apo A-I_(unox). In preferred embodiments, rHDL-2 arereconstituted HDL particles containing only apo A-I_(ox). In preferredembodiments, rHDL-3 are reconstituted HDL particles containing apoA-I_(unox) and apo A-I_(ox) with a molar ratio of 1:1. In preferredembodiments, rHDL-4 are reconstituted HDL particles containing apoA-I_(unox), apo A-I_(ox) and apo A-II_(unox) with a molar ratio of3:3:1.

In preferred embodiments, apo A-I, apo A-II or fragments thereof arefirst chemically or enzymatically modified and then the syntheticnanoparticle of the invention is assembled using this modifiedapolipoprotein. It might be possible, however, to selectively modifyonly apolipoprotein portion of the fully assembled syntheticnanoparticle of the invention.

The metallic contrast agent may preferably be selected from the groupconsisting of Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Fe(III), Pr(III), Nd(III) Sm(III), Tb(III), Yb(III) Dy(III), Ho(III),Eu(II), Eu(III), and Er(III), Tl²⁰¹, K⁴², In¹¹¹, Fe.⁵⁹, Tc^(99m), Cr⁵¹,Ga⁶⁷, Ga⁶⁸, Cu⁶⁴, Rb⁸², Mo⁹⁹, Dy¹⁶⁵. In addition, the metallic contrastagents could include crystals and other particulate materials (oxides,quantum dots, etc.) The non-metallic contrast agent may preferably beselected from the group consisting of Fluorescein, Carboxyfluorescein,Calcein, F¹⁸, Xe¹³³, I¹²⁵, I¹³¹, I¹²³, P³², C¹¹, N¹³, O¹⁵, Br⁷⁶, Kr⁸¹.In specific embodiments, the metallic contrast agent is gadolinium. Thenon-metallic contrast agent may still preferably be selected from thegroup of iodinated contrast media consisting of ionic monomers anddimers, and nonionic monomers and dimers, including, but not limitingto, Diatrizoate, Metrizoate, Isopaque, Ioxaglate, Iopamidol, Iohexol,and Iodixanol (Singh J. & Daftary A. J Nucl Med Technol 2008; 36:69-74;Stacul F. Eur Radiol 2001; 11:690-7). In particularly preferredembodiments, the metallic or non-metallic contrast agent is conjugatedto a lipid component of the synthetic nanoparticle. Such a lipidcomponent of the synthetic nanoparticle may selected from the groupconsisting of a sterol, a phospholipid, a sterol ester, a diacylglyceroland a triacylglycerol. In preferred embodiments the non-metalliccontrast agent is1-palmitoyl-2-((E)-10,11-diiodo-undec-10-enoyl)-sn-glycero-3-phosphocholine.In certain embodiments, the sterol of the imaging agent is cholesterol.In other embodiments, the sterol ester is cholesteryl ester. In stillpreferred embodiments, the metallic or non-metallic contrast agent isencapsulated into the synthetic nanoparticle.

In preferred embodiments, the metallic or non-metallic contrast agent isassociated with a phospholipid and preferably, selected from the groupconsisting of phosphatidylcholine (PC), phosphatidylethanolamine (PE),phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol(PG), cardiolipin (CL), a sphinolipid, sphingomyelin (SM), andphosphatidic acid (PA). In particularly preferred embodiments, thephospholipid is PC. The PC (or indeed any other phospholipids) maycomprise any fatty acid of e.g., between 4 and 24 carbon chains inlength. The two fatty acids of the phospholipids may be the same or theymay be different. In preferred embodiments, the PC is POPC. In yet otherpreferred embodiments, the phospholipid is PE. In specific embodiments,the phospholipid is dimyristoyl-PE (DMPE). In still other preferredembodiments, the phospholipid is a modified PE. Preferably, the modifiedPE is poly-lysine PE. In still other embodiments, the poly-lysine PE ispoly-lysine dimyristoyl-PE.

It is contemplated that the metallic or non-metallic contrast agent mayalternatively be conjugated to a modified protein component of thesynthetic nanoparticle. Such a modified protein may be selected from thegroup consisting of a modified apo A-I or fragments thereof, a modifiedA-II or fragments thereof, and any combination of a modified apo A-I anda modified A-II and fragments thereof.

According to the present invention, a high density lipoprotein (HDL),e.g. nascent HDL, reconstituted HDL (rHDL), recombinant HDL or anHDL-like particle is particularly preferred which has a molar ratio of amodified apolipoprotein (selected the group consisting of a modified apoA-I or fragments thereof, a modified A-II or fragments thereof, and anycombination of a modified apo A-I and a modified A-II and fragmentsthereof) and phospholipid in the range of 1:50 to 1:250, particularlyabout 1:150. Further, rHDL may optionally contain additional lipids suchas cholesterol, cholesterol esters, triglycerides and/or sphingolipids,preferably in a molar ratio of up to 1:20, e.g. 1:5 to 1:20 based on theapolipoprotein. Preferred rHDL is described in Eur Pat EP-A-0663 407.Further, HDL-like particle may optionally contain1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and cholesterol.

Production of reconstituted lipoprotein nanoparticles such asreconstituted high-density lipoproteins (rHDL) and other HDL-likenanoparticles is described, by way of example, in (US Pat Appl20060217312; US Pat Appl 20060205643; U.S. Pat. No. 5,652,339; Lerch etal. Vox Sang 1996; 71:155-64; Matz C. E. & Jonas A. J Biol Chem 1982;257:4535-40; Toledo et al. Arch Biochem Biophys 2000; 380:63-70; SigalovA. B. & Stern L. J. Chem Phys Lipids 2001; 113:133-46; Kim et al.Journal of Hepatology 2009; 50:479-88; US Pat Appl 2009/0312402; U.S.Pat. No. 6,008,202; Mukundan et al. AJR Am J Roentgenol 2006; 186:300-7;U.S. Pat. No. 7,588,751). Production of recombinant HDL is described, byway of example, in Eur Pat EP 469017 (in yeast), U.S. Pat. No. 6,559,284(in E. coli), and WO 87/02062 (in E. coli, yeast and Cho cells) and WO88/03166 (in E. coli). The contents of each of these documents areincorporated herein by reference. Preferably, the HDL is reconstitutedHDL. In some aspects of the present invention, rHDL may be prepared froma modified apolipoprotein (selected the group consisting of a modifiedapo A-I or fragments thereof, a modified A-II or fragments thereof, andany combination of a modified apo A-I and a modified A-II and fragmentsthereof), and soybean-derived PC, mixed in molar ratios of approximately1:150 apolipoprotein:PC.

In preferred embodiments, the synthetic nanoparticle in the imagingagent comprises a phospholipid:sterol:apolipoprotein ratio of 180:5:3(mol:mol:mol). In other preferred embodiments, the syntheticnanoparticle in the imaging agent comprises aphospholipid:apolipoprotein ratio of 100:3 (mol:mol). In still preferredembodiments, the synthetic nanoparticle in the imaging agent comprises aphospholipid:steryl ester:sterol:triglycerides (TG):apo A-I ratio (w/w)of 100:62:25:11:2. In other preferred embodiments, the syntheticnanoparticle comprises a DOTAP:cholesterol ratio of 1:1 (mol:mol) and alipid/apo A-I protein ratio of 10:1 (w/w).

In the imaging agents of the invention, at least one apolipoproteinmolecule (or fragment thereof) per synthetic nanoparticle is modified.In specific embodiments, the imaging agent comprises between 1 and 50metallic or non-metallic contrast agent molecules per syntheticnanoparticle. In various aspects of the invention, the metallic ornon-metallic contrast agent molecule is conjugated to a phospholipidmoiety and the phospholipid moiety accommodates more than one metallicor non-metallic agent molecule. In more specific aspects, the imagingagent comprises 10 metallic or non-metallic contrast agent molecules persynthetic nanoparticle. The imaging agent of the invention may comprisebetween about 80 and about 180 phospholipids per synthetic nanoparticle.Other embodiments define the imaging agent as comprising 2, 3 or 4apolipoprotein molecules per synthetic nanoparticle. In still furtherembodiments, the synthetic nanoparticle comprises 1 apolipoproteinmolecule to between about 30 and about 60 phospholipid molecules.

The imaging agents of the invention may further comprise an additionaltargeting moiety to further facilitate targeting of the agent to aspecific site in vivo. The additional targeting moiety may be any moietythat is conventionally used to target an agent to a given in vivo siteand may include but is not limited to, an antibody, a receptor, aligand, a peptidomimetic agent, an aptamer, a polysaccharide, a drug anda product of phage display. In particular embodiments, the targetingmoiety may be conjugated to a detectable label. For example, apoE-derived lipopeptide (Chen et al. Contrast Media Mol Imaging 2008;3:233-42), an apo A-I mimetic peptide (Cormode et al. Small 2008;4:1437-44), murine (MDA2 and E06) or human (IK17) antibodies that bindunique oxidation-specific epitopes (Briley-Saebo et al. Circulation2008; 117:3206-15), USPIO particles (US Pat Appl 2009/0004113), and goldparticles (Cormoe et al. Radiology 2010; 256:774-82) may be used in thepresent invention to further improve specific targeting macrophages,decrease the required dosage of administered contrast agents including,but not limiting to, Gd-based contrast agents, and increase an imagequality of vulnerable plaques.

Preferably, the diameter or the longest dimension of the nanoparticle isbetween about 5 nm to about 18 nm. The diameter may be between about 5to about 12 nm. In particularly preferred embodiments, the diameter isless than 10 nm. In some embodiments the diameter is more than 100 mm.

The imaging agent may be one which comprises two or more differentcontrast agents. These two or more different contrast agents may all bemetallic, all be non-metallic or the imaging agent may comprise somemetallic contrast agents and some non-metallic contrast agents. Inadditional embodiments, the agent may further comprise a drug to bedelivered at an in vivo site targeted by the targeting moiety.

Other aspects of the present application describe a pharmaceuticalcomposition comprising an imaging agent as described herein and apharmaceutically acceptable carrier or diluent.

Also contemplated are methods of in vivo imaging of a site within asubject comprising administering to the subject an imaging agentcomprising a metallic or non-metallic contrast agent conjugated tocomponent of a synthetic nanoparticle. The imaging agent of theinvention used in such a method comprises a targeting moiety thatspecifically targets the imaging agent to the in vivo site. In specificembodiments, the in vivo site being imaged is the site of anatherosclerotic plaque. In specific embodiments, an additional targetingmoiety is selected from the group consisting of antibodies againstlipoprotein lipase, oxidized epitopes on atherosclerotic plaques oxLDLMDA, antibodies against matrix metalloproteinases and anti-tissue factorantibodies.

In other embodiments, the in vivo site is the site of a tumor and theadditional targeting moiety comprises a moiety that recognizes atumor-specific binding partner present on the tumor. More particularly,the binding partner is selected from the group consisting of an antibodyagainst a tumor-specific antigen, a receptor for a ligand expressed bythe tumor, a ligand for a receptor expressed on the tumor.

Further aspects of the invention are directed to methods of making animaging composition, the method comprising: obtaining a compositioncomprising a phospholipid optionally covalently linked to a chelatingmoiety and reacting the composition with a composition comprising afirst metallic or non-metallic contrast agent to produce aphospholipid-chelating (optionally) agent-metallic/non-metallic agentconjugate; co-sonicating the conjugate of step (a) with: a predeterminedamount of HDL apolipoprotein; a predetermined amount of a mixture ofphospholipids mixed in a ratio found in circulating HDL; a predeterminedamount of sterol; and a predetermined amount of HDL core lipidscomprising triglycerides (TAG) and cholesteryl ester in a ratio found incirculating HDL; for a time period sufficient to allow the conjugate andthe individual components of nanoparticle to coalesce intonanoparticulate structures; and isolating structures that have a size ofbetween about 5 to about 12 nm diameter. In preferred embodiments, thefirst metallic contrast agent is selected from the group consisting ofGd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Fe (III), Pr(III),Nd(III) Sm(III), Tb(III), Yb(III) Dy(III), Ho(III), Eu(II), Eu(III), andEr(III), Tl²⁰¹, K⁴², In¹¹¹, Fe.⁵⁹, Tc^(99m), Cr⁵¹, Ga⁶⁷, Ga⁶⁸, Cu⁶⁴,Rb⁸², Mo⁹⁹, Dy¹⁶⁵. The first non-metallic contrast agent is selectedfrom the group consisting of Fluorescein, Carboxyfluorescein, Calcein,F¹⁸, Xe¹³³, I¹²⁵, I¹³¹, I¹²³, P³², C¹¹, N¹³, O¹⁵, Br⁷⁶, Kr⁸¹. Thenon-metallic contrast agent may still preferably be selected from thegroup of iodinated contrast media consisting of ionic monomers anddimers, and nonionic monomers and dimers, including, but not limitingto, Diatrizoate, Metrizoate, Isopaque, Ioxaglate, Iopamidol, Iohexol,and Iodixanol (Singh J. & Daftary A. J Nucl Med Technol 2008; 36:69-74;Stacul F. Eur Radiol 2001; 11:690-7).

The methods of the making the compositions may further compriseproviding a second phospholipid-chelating agent-metallic or non-metalliccontrast agent, wherein the second contrast agent is different from thefirst contrast agent. In exemplary embodiments, one of the contrastagents may be a metallic agent whereas the second contrast agent is anon-metallic agent. In those embodiments that employ a chelating agent,the chelating agent preferably is selected from the group consisting ofDTPA, EDTA, BOPTA, DOTA, DO3A and aDO3A. In preferred embodiments, thesterol component is selected from the group consisting of cholesterol,stigmasterol, ergosterol, lanosterol, and sitosterol. In other preferredembodiments, the phospholipid in the phospholipid-chelatingagent-metallic (or non-metallic) contrast agent conjugate is selectedfrom the group consisting of consisting of PC, PE, PS, PI, PG, CL, SMand PA. Preferably, the phospholipid mixture is a mixture of two or morephospholipids selected from the group consisting of consisting of PC,PE, PS, PI, PG, CL, SM and PA.

In preferred embodiments, the phospholipids, sterol, and apolipoproteinare mixed in a phospholipid:sterol:apolipoprotein ratio of 180:5:3(mol:mol:mol). In other preferred embodiments, the phospholipids andapolipoprotein are mixed in a phospholipid:apolipoprotein ratio of 100:3(mol:mol). In other preferred embodiments, the phospholipids, corelipids, sterol, and apolipoprotein are mixed in a phospholipids:sterylester:sterol:TG:apo A-I (w/w) of 100:62:25:11:2. In preferredembodiments, the method produces reconstituted synthetic nanoparticleparticles that comprise between about 80 and about 180 phospholipids persynthetic nanoparticle. Preferably, the method produces a lipoproteinnanoparticle that comprise 2, 3 or 4 apolipoprotein molecules at leastone of which is modified per synthetic particle. Still preferably, themethod produces nanoparticle that comprises 1 apolipoprotein molecule tobetween about 30 and about 60 phospholipid molecules. The methods areused to produce synthetic nanoparticle that comprise between 1 and 30metallic contrast ions per synthetic nanoparticle. In specificembodiments, the phospholipid covalently linked to a chelating moiety isa modified phospholipid that can accommodate more than one metallic ornon-metallic contrast agent. Preferably, the modified phospholipid is apoly-L-lysine-PE. Still more preferably, the poly-L-lysine-PE isdimyristoyl-poly-L-lysine. In preferred aspects the method furthercomprises obtaining a composition comprising a biotinylated phospholipidreacting the composition with a composition comprising an additionaltargeting agent to produce a phospholipid-targeting agent conjugate, andproviding the phospholipid-targeting agent conjugate in theco-sonicating mixture.

In still preferred embodiments, a typical liposomal HDL-like compositioncomprises a lipid or phospholipid, a stabilizing excipient such ascholesterol, a polymer-derivatized phospholipid, and apolipoprotein.Suitable examples of lipids or phospholipids, stabilizing excipients,and polymer-derivatized phospholipids are set forth in, for example, USPat Appls 20090311191 and 20100202974, all of which are incorporated byreference in their entireties herein. The liposomal HDL-likecompositions typically encapsulate or associate a contrast agent. Itshould be noted that for purposes of the present application, theidentity of the contrast agent is not of substantial importance. Rather,the modified apolipoprotein, the liposome composition (e.g.,cholesterol; at least one phospholipid; and at least one phospholipidwhich is derivatized with a polymer chain) and the small size (e.g.,less than 150 nm, as described below) provide the desired localization.In some aspects of the present invention, the liposomal HDL-likecompositions may be prepared from a modified apolipoprotein selected thegroup consisting of a modified apo A-I or fragments thereof, a modifiedA-II or fragments thereof, and any combination of a modified apo A-I anda modified A-II and fragments thereof. Nonetheless, suitable contrastagents include, for example, fluorescent dyes, such as, for example,fluorescein iso-thiocynate (FITC) and rhodamine; CT contrast agentsincluding iodinated compounds such as iohexol, iodixanol, and iotrolan,and as otherwise described in US Pat Appl 20100202974, US Pat Appl20090311191, U.S. Pat. No. 7,588,751, and U.S. patent application Ser.Nos. 10/830,190, 11/595,808, and 11/568,936; and MRI contrast agentsincluding lanthanide aminocarboxylate complexes such as Gadolinium (III)DTPA, Gd-DOTA, Gd-DOTAP, and Gd-DOTMA.

Also contemplated herein are diagnostic kits comprising a metallic ornon-metallic contrast agent conjugated to component of the compositionof the invention in a pharmaceutically acceptable carrier or diluent;and a device for delivering the composition to a subject prior todiagnostic imaging of the subject. Other kits contemplated herein arekits for producing an imaging agent the kit comprising a firstcomposition comprising a metallic or non-metallic contrast agent; asecond composition comprising a phospholipid covalently linked to achelating moiety; a third composition comprising apolipoproteins A-I andA-II; and a fourth composition comprising free phospholipid. Inaddition, the kits may comprise instructions for reconstituting HDL.Preferred metallic agents for the first composition include but are notlimited to, Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Fe (III),Pr(III), Nd(III) Sm(III), Tb(III), Yb(III) Dy(III), Ho(III), Eu(II),Eu(III), and Er(III), Tl²⁰¹, K⁴², In¹¹¹, Fe.⁵⁹, Tc^(99m), Cr⁵¹, Ga⁶⁷,Ga⁶⁸, Cu⁶⁴, Rb⁸²,Mo⁹⁹, Dy¹⁶⁵. In addition, the metallic contrast agentsinclude crystals and other particulate materials (oxides, quantum dots,etc.). Preferred non-metallic agents for the first composition includebut are not limited to Fluorescein, Carboxyfluorescein, Calcein, F¹⁸,Xe¹³³, I¹²⁵, I³¹, I¹²³, P³², C¹¹, N¹³, O¹⁵, Br⁷⁶, Kr⁸¹. The non-metalliccontrast agent for the first composition may still preferably beselected from the group of iodinated contrast media consisting of ionicmonomers and dimers, and nonionic monomers and dimers, including, butnot limiting to, Diatrizoate, Metrizoate, Isopaque, Ioxaglate,Iopamidol, Iohexol, and Iodixanol (Singh J. & Daftary A. J Nucl MedTechnol 2008; 36:69-74; Stacul F. Eur Radiol 2001; 11:690-7).

Any contrast agent that is employed in MRI, CT, Gamma-scintigraphy, oroptical imaging techniques may be used in the present invention. Thekits may further comprise a fifth composition comprising sterol, such ase.g., cholesterol, stigmasterol, ergosterol, lanosterol, and sitosterol.The kits may further comprise a sixth composition comprising a HDL corelipids. In particular aspects, the fourth composition comprising thephospholipid comprises either individually or as a mixture one or morephospholipids selected from the group consisting of consisting of PC,PE, PS, PI, PG, CL, SM and PA. In certain embodiments, the phospholipidsis covalently linked to a chelating moiety such as e.g., DTPA, EDTA,BOPTA, DOTA, DO3A and aDO3A.

Preferably, the second phospholipid composition is a poly-L-lysine-PE.More particularly, poly-L-lysine-PE is poly-L-lysine-DMPE. As usedherein the “core lipids” are those lipids that form the core of thenanoparticle. Preferably, the core lipids comprise cholesteryl esterand/or TG.

The present invention addresses this need by providing reconstitutedhigh density lipoprotein (rHDL) particles and liposomal HDL-likecompositions that contain modified apolipoproteins or fragments thereof(FIGS. 1, 2A, and 2B). The particles of the invention are used fortargeted delivery of imaging contrast agents conjugated to a component(or components) of rHDL to sites of interest. By “rHDL” it should beunderstood that the present invention contemplates a synthetic moleculethat exhibits some of the characteristics and features of plasma-derivedHDL moieties (e.g., small diameter, non-atherogenic, ability to promotecholesterol efflux, stability, etc.) but is a synthetic molecule and isnot formed from plasma HDL. Commercial sources of HDL include: BiodesignInternational; Athens Research and Technology; Intracel Corp; ScrippsLaboratories; Academy Biomedical Co; CSL Behring AG, Pfizer, Inc. Thus,the compositions of the present invention comprise a contrastagent-containing synthetic rHDL moiety, major protein constituents ofwhich, apolipoproteins, or fragments thereof are modified in a means forconverting them into targeting moieties (for example, into substratesfor macrophages) for targeted delivery of rHDL-associated contrastagents to sites of interest (for example, an atherosclerotic plaque).The use of synthetic rHDL containing modified apolipoproteins orfragments thereof for imaging in vivo sites is advantageous becauseafter administration, most of these particles get bound and/or uptakenby cells at sites of interest (FIGS. 3A and 3B). This allows asignificant reduction in the contrast agent dosage required and therebylimits concerns related to systemic toxicity reduce the contrast agentdosage required and thus limit concerns related to systemic toxicity,which is especially important for Gd-based contrast agents.

Native HDL that contain unmodified lipids and apolipoproteins are notrecognized by macrophage scavenger receptors (Platt N. and Gordon S. JClin Invest 2001; 108:649-54). As a result, native HDL do notirreversibly bind to macrophages and are not uptaken by macrophages. Incontrast, modified (for example, oxidized) HDL are readily absorbed bymacrophages resulting to accumulation of the modified HDL and theircomponents such in macrophage-enriched atherosclerotic plaques (Bergt etal. Eur J Biochem 2001; 268:3523-31; Bergt et al. Biochem J 200; 346 Pt2:345-54; Bergt et al. Arterioscler Thromb Vasc Biol 2003; 23:1488-90;Bergt et al. Proc Natl Acad Sci USA 2004; 101:13032-7; Bergt et al. FEBSLett 1999; 452:295-300; Pankhurst et al. J Lipid Res 2003; 44:349-55;Panzenboeck et al. J Biol Chem 1997; 272:29711-20). Further, oxidationof native HDL by a natively occurring oxidative system, themyeloperoxidase/hydrogen peroxide/chloride system, results in oxidativemodification of both lipid and protein constituents of HDL (Panzenboecket al. J Biol Chem 1997; 272:29711-20; Bergt et al. Eur J Biochem 2001;268:3523-31). In line with this, both oxidized lipids and oxidativelydamaged apolipoprotein A-I molecules such as apolipoprotein A-I₊₃₂(containing two sulfoxidized methionine residues) and apolipoproteinmoieties containing 3-Chloro- or 3-Nitrotyrosine residue at position 192have been found in atherosclerotic plaques (Pankhurst et al. J Lipid Res2003; 44:349-55; Bergt et al. Proc Natl Acad Sci USA 2004; 101:13032-7;Hazen S. L. & Heinecke J. W. J Clin Invest 1997; 99:2075-81; Hazen etal. Circ Res 1999; 85:950-8; Malle et al. Biochem Biophys Res Commun2001; 289:894-900). As described herein, it is unexpectedly found thatoxidative modification of only protein constituents or peptide fragmentsthereof of rHDL is sufficient to convert these particles to substratesfor macrophage scavenger receptors and to result therefore in theimprovement of association of Gd-(HDL/modified apolipoprotein)-particlewith macrophages and/or absorption (uptake) of Gd-(HDL/modifiedapolipoprotein)-particle by macrophages when compared to that of theGd-(HDL/apolipoprotein)-particle constructed with non-modified naturallyoccurring apo A-I, apo A-II or fragments thereof. Compositions of theinvention, rHDL, contain certain chemical or enzymatic modification ofapolipoproteins (apo) A-I or A-II or fragments thereof and can be easilyand reproducibly produced. These advantageous compositions aredemonstrated by the present invention to solve numerous problems whichotherwise are associated with high dosages of Gd required and the lackof control and reproducibility of formulations, especially inlarge-scale production.

In addition, the particles of the present invention have all other knownadvantages of synthetic rHDL conjugated to contrast agents (US Pat Appl20070243136): first, of all of the lipoprotein compositions, rHDL arethe easiest to reproducibly reconstitute, and they are sufficientlysmall (about 10 nm diameter) (Rensen et al. Adv Drug Deliv Rev 2001;47:251-76) to penetrate readily into the extracellular space and freelyenter and exit sites such as the sites of atherosclerotic plaques (Sloopet al. J Lipid Res 1987; 28:225-37; O'Brien et al. Circulation 1998; 98,519-27; Kunjathoor et al. Arterioscler Thromb Vase Biol 2002; 22:462-8).Second, rHDL has the advantage of not being atherogenic and, therefore,will not pose the same cardiovascular risks that might be associatedwith the use of LDL moieties. Moreover, unlike LDL moieties andmicelles, the HDL moieties are not retained at the in vivo site forprolonged periods of time.

The preferred particles of the invention comprise at least one modifiedapolipoprotein A-I and/or A-II or peptide fragments thereof and at leastone amphipathic lipid, to form a structure that can be spherical ordiscoidal. To readily penetrate into the interstitial fluid, theparticles must be 25 nm or less in diameter, if spherical, or 25 nm orless in their longest dimension, if discoidal. For structural stability,ease of manufacture, and ability to carry significant amounts ofcontrast and/or targeting agents, the particles should be at least 5 nmin their largest dimension. Furthermore, to carry large amounts of acontrast agent, said agent is incorporated through the inclusion of anamphipathic chelator, that is, a molecule that has a hydrophobic portionthat incorporates into the particle, and a chelating portion that bindsan MRI contrast agent.

The inclusion of an amphipathic protein or peptide aids the structuralstability of the particle, particularly when the particle has adiscoidal shape. Exemplary proteins or peptides are selected from themajor protein constituents of HDL, an apolipoprotein A-I, anapolipoprotein A-II, and peptide fragments thereof. Apolipoproteins thatmay be used herein are commercially available from e.g., BiodesignInternational; Athens Research and Technology; Intracel Corp; ScrippsLaboratories; Academy Biomedical Co). Alternatively, standard proceduresthat are well known in the art can be used to isolate and purifyapolipoproteins A-I and A-II from human serum (Sigalov et al. JChromatogr 1991; 537, 464-8). One skilled in the art, using the knownprimary sequences of apolipoproteins A-I and A-II, can easily synthesizethe peptide fragments of this invention. Standard procedures forpreparing synthetic peptides are well known in the art. The fragmentscan be synthesized using the solid phase peptide synthesis method ofMerrifield (Merrifield, R. B. Biochemistry 1964; 3:1385-90), which isincorporated herein by reference) or modifications of this method. Theinclusion of an amphipathic lipid creates a hydrophobic zone within theparticle that allows the incorporation of an amphipathic chelator orother attaching agent. Lipids that may be used herein are commerciallyavailable from e.g., Avanti Polar Lipids.

The hydrophobic zone also allows the incorporation of an amphipathiccomplex that comprises an additional targeting agent. Said complexesinclude, but are not limited to, an amphipathic lipid covalently linkedto an antibody; and an amphipathic lipid covalently linked to an avidin,which is then non-covalently linked to a biotinylated antibody. Othercomplexes include polylysine phospholipids-Gd complexes in which theadditional targeting moiety is provided. Other complexes include1-palmitoyl-2-((E)-10,11-diiodo-undec-10-enoyl)-sn-glycero-3-phosphocholineas described in (Elrod et al. Nanomedicine: Nanotech, Biol and Med 2009;5:42-5).

Apolipoproteins and fragments thereof of the present invention must bemodified first and then used (alone or in combination with unmodifiedapolipoprotein molecules and fragments thereof) for reconstitution ofrHDL particles rather than modified in the context of assembled HDLparticles. The use of preliminary modified apolipoproteins and fragmentsthereof of the invention is advantageous for several reason. Firstly, itis well known in the art that modification, for example, of lipid-freeand lipid-bound apolipoprotein A-I affects discrete regions of theproteins and cannot be controlled for lipid-bound protein (Bergt et al.Biochem J 200; 346 Pt 2:345-54). Secondly, modified lipid-free proteincan be easily purified and well characterized using standard procedureswell known in the art. Thirdly, having only one molecule of modifiedapolipoprotein A-I, modified apolipoprotein A-II, or modified fragmentsthereof of the present invention per rHDL particle is sufficient totarget this particle to sites of interests, for example, to macrophagesof atherosclerotic plaques. Thus, the structural stability of theparticle can be provided by other, unmodified apolipoprotein molecules,minimizing any potential side effects of modified proteins of theinvention. Exemplary methods of modifying and purifying theapolipoproteins and fragments thereof of the invention are described infurther detail in the examples herein below.

Finally, for several reasons, the rHDL particles must be syntheticrather than isolated from human plasma. In particular, the use ofnaturally occurring lipoproteins isolated from human plasma is notcontemplated, rather the rHDL particles described herein are synthetic.The use of synthetic molecules have a number of potential advantages.Firstly, the use of such molecules avoids transmission of blood-borneinfectious agents, which are frequently present in conventionallyisolated human plasma lipoproteins. Secondly, isolation of largequantities of human plasma lipoproteins is impractical and expensive,because it requires large amounts of human plasma as starting materialand then tedious isolation procedures. Thirdly, human plasmalipoproteins vary substantially from batch to batch, depending on theindividual donor(s), recent dietary intake, and other factors. Fourthly,synthetic particles allows the skilled artisan to circumvent theproblems of oxidized or readily oxidizable lipids, such as highlyunsaturated lipids. Oxidized or readily oxidizable lipids can be toxicand can impair stability during storage. As noted elsewhere, thepreferred amphipathic lipid is POPC, which is relatively resistant tooxidation and is readily available commercially in pure form. Fifthly,incorporation of large amounts of contrast agents and/or targetingagents into a pre-existing lipoprotein is difficult, if not impossible,without significant disruption of said pre-existing lipoprotein.Exemplary methods of forming the synthetic rHDL compositions of theinvention using modified apolipoproteins and fragments thereof of theinvention are described in further detail in the examples herein below.

In particularly preferred aspects of the invention the synthetic rHDLare reconstituted with the contrast agent, modified apolipoproteins andfragments thereof, and a second agent that allows the additionaltargeting of the imaging composition to a specific site. While some ofthe discussion herein focuses on atherosclerotic plaques, it should beunderstood that other sites in the body also may be targeted with thecompositions of the invention. These compositions of the invention areable to locate to a specific target site and produce a desirable resultof a large increase in signal intensity from plaque retention of thesynthetic rHDL because of the presence of a specific targeting moleculeof interest. This is of particular interest because it has previouslybeen noted that, for example, in atherosclerotic plaques HDL is veryinefficiently retained in the plaque site. Although it may be possibleto increase the retention of HDL moiety by increasing the relativeamount of apo E or C-reactive protein present in the HDL, it iscontemplated that the presence of the targeting moieties comprised ofchemically or enzymatically modified apolipoproteins or fragmentsthereof is advantageous because it facilitates the controlled retentionof the imaging agent at the site of interest, thereby promoting anincrease in the signal at that site. The presence of an additionaltargeting moiety can further facilitate the controlled retention of theimaging agent at the site of interest, thereby further promoting anincrease in the signal at that site as compared to use of the modifiedapolipoproteins and fragments thereof alone.

As described herein it is unexpectedly found that the targeted MRIcontrast compositions can be formulated as conjugates of a component ofa synthetic rHDL composition comprised of chemically or enzymaticallymodified apolipoproteins or fragments thereof of the present invention.These metal imaging agents are useful in all areas of diagnostics thatcan employ MRI imaging of a given tissue or in vivo site. In exemplaryembodiments, the compositions of the present invention are generated byincorporating a paramagnetic metal ion complexed with a chelating agenthaving a lipophilic moiety, into synthetic rHDL moieties of theinvention. Preferably, the chelating agent is a polyaminopolycarboxylatechelating agent. Further, it is preferred that there are multiple metalions complexed per synthetic rHDL entity. The lipophilic paramagneticchelate that serves as the metallic contrast agent will preferably beconjugated to a phospholipid moiety. As phospholipids are an integralare relatively easily incorporated into synthetic rHDL moieties, and sothe metal contrast agent is thus easily incorporated into the syntheticrHDL compositions of the present invention. The entire complex whichcontains the HDL reconstituted using modified apolipoproteins andfragments thereof as targeting moieties and the metal contrast agent,and optionally also contains an additional targeting moiety, is referredto herein as an “imaging agent.”

Advantageously, complexing the metallic contrast agent in the mannerdescribed herein eliminates or reduces the toxicity and otherundesirable side effects of the metallic agent whilst at the same timeretaining the paramagnetic properties of the metal ion that confer theimaging action of the paramagnetic ion, i.e., change in relaxivity ofthe hydrogen atoms of water. As discussed herein below, numerouschelating agents may be used to produce a paramagnetic ion compositionthat would be suitable for reconstitution in synthetic rHDL compositionsherein. However, it is envisioned that polyaminopolycarboxylic acidswill be particularly useful for complexing the paramagnetic ionsintended for MRI imaging of human or animal body.

Methods and compositions for making and using the imaging agents of thepresent invention are described in further detail herein below. Asdescribed herein it was unexpectedly found that the MRI contrastcompositions formulated as conjugates of a component of a synthetic rHDLcomposition (US Pat Appl 20070243136) can be specifically targeted tosites of interest by chemical or enzymatic modification of the majorprotein constituents of HDL, apolipoproteins A-I and A-II, or fragmentsthereof, solving therefore numerous problems which otherwise areassociated with high dosages of Gd required and the lack of control andreproducibility of formulations, especially in large-scale production.These metal imaging agents are useful in all areas of diagnostics thatcan employ MRI imaging of a given tissue or in vivo site.

A. Apolipoproteins and Apolipoprotein Peptides

In the methods of the present invention, the lipoproteins of interestare HDLs and their synthetic reconstituted analogues. The functionalcharacteristics of HDL particles are mainly determined by their majorapolipoprotein (apo) components such as apo A-I and A-II. Each HDLparticle usually comprises at least 1 molecule, and usually two to 4molecules, of apo A-I. Apo A-I is synthesized by the liver and smallintestine as preproapolipoprotein A-I, which is secreted asproapolipoprotein A-I (proApoA-I) and rapidly cleaved to generate theplasma form of ApoA-I, a single polypeptide chain of 243 amino acids(Brewer et al. Biochem Biophys Res Commun 1978; 80:623-30). Apo A-Icomprises 6 to 8 different 22-amino acid alpha-helices or functionalrepeats spaced by a linker moiety that is frequently proline. The repeatunits exist in amphipathic helical conformation (Segrest et al. FEBSLett 1974; 38:247-58) and confer the main biological activities of apoA-I, i.e., lipid binding and lecithin cholesterol acyl transferase(LCAT) activation. Apo A-I plays an important role in lipid transportand metabolism. It promotes cholesterol efflux (Chambenoit et al. J BiolChem 2001; 276:9955-60; Rothblat et al. J Lipid Res 1992; 33:1091-7),acts as a cofactor for the LCAT enzyme (Jonas et al. Biochim BiophysActa 1993; 1166:202-10; Frank et al. Biochemistry 1998; 37:13902-9) andas a ligand that binds to the class B scavenger receptor SR-BI (Acton etal. Science 1996; 271:518-20). Apo A-I shows endotoxin neutralization(Massamiri et al. J Lipid Res 1997; 38:516-25) and also protects againstthe cytotoxic effects of oxidized LDL (Suc et al. Arterioscler ThrombVasc Biol 1997; 17:2158-66).

The nature of the apolipoproteins comprising the apolipoprotein fractionof the compositions of the present invention is not critical forsuccess. Examples of suitable apolipoproteins include, but are notlimited to, preproapolipoprotein forms of apoA-I and apoA-II; pro- andmature forms of human apoA-I and apoA-II; and active polymorphic forms,isoforms, variants and mutants as well as truncated forms, the mostcommon of which are apoA-I_(Milano) and apoA-I_(P), as disclosed in USPat Appl 20060217312, the disclosure of which is incorporated herein byreference. Apolipoproteins mutants containing cysteine residues are alsoknown, and can also be used (see, e.g., US Pat Appl 20030181372). Theapolipoproteins may be in the form of monomers or dimers, which may behomodimers or heterodimers. For example, homo- and heterodimers (wherefeasible) of pro- and mature apoA-I (Duverger et al. Arterioscler ThrombVasc Biol 1996; 16:1424-9), apoA-I_(Milano) (Franceschini et al. J BiolChem 1985; 260:16321-5), apoA-I_(P) (Daum et al. J Mol Med 1999;77:614-22), and apoA-II (Shelness G. S. & Williams D. L. J Biol Chem1984; 259:9929-35; Shelness G. S. & Williams D. L. J Biol Chem 1985;260:8637-46) may be used. The apolipoproteins may include residuescorresponding to elements that facilitate their isolation, such as Histags, or other elements designed for other purposes, so long as theapolipoprotein retains some biological activity when included in acomplex.

Such apolipoproteins can be purified from animal sources (and inparticular from human sources) or produced recombinantly as iswell-known in the art (see, e.g., Sigalov et al. J Chromatogr 1991;537:464-8; Chung et al. J Lipid Res 1980; 21:284-91; Cheung et al. JLipid Res 1987; 28:913-29; see also U.S. Pat. Nos. 5,059,528, 5,128,318,6,617,134, and U.S. Pat Appls 20020156007, 20040067873, 20040077541, and20040266660).

Non-limiting examples of peptides and peptide analogs that correspond toapoA-I, apoA-I_(Milano), and apoA-II, and are suitable for enzymaticand/or chemical modifications and subsequent use as peptide fragments ofmodified apolipoproteins in the complexes and compositions describedherein are disclosed in U.S. Pat. Nos. 6,004,925, 6,037,323 and6,046,166, 5,840,688, US Appls 20040266671, 20040254120, 20030171277,20030045460, 20030087819, and 20060217312, the disclosures of which areincorporated herein by reference in their entities. These peptides andpeptide analogues can be composed of L-amino acid or D-amino acids ormixture of L- and D-amino acids. They may also include one or morenon-peptide or amide linkages, such as one or more well-knownpeptide/amide isosteres. Such “peptide and/or peptide mimetic”apolipoproteins can be synthesized or manufactured using any techniquefor peptide synthesis known in the art, including, e.g., the techniquesdescribed in U.S. Pat. Nos. 6,004,925, 6,037,323 and 6,046,166.

The complexes and compositions of the present invention may include asingle type of apolipoprotein and/or peptide fragments thereof, ormixtures of two different apolipoproteins and peptide fragments thereof,which may be derived from the same or different species. Although notrequired, the charged lipoprotein complexes will preferably compriseapolipoproteins that are derived from, or correspond in amino acidsequence to, the animal species being treated, in order to avoidinducing an immune response to the therapy. The use of peptide mimeticapolipoproteins may also reduce or avoid an immune response.

B. Modified Apolipoproteins and Apolipoprotein Peptides

Free radicals, oxidative stress, and antioxidants have become commonlyused terms in modern discussions of disease mechanisms. Oxidativemodifications of proteins plays a crucial role in aging and otherphysiological processes because oxidized proteins lose catalyticfunction (Sohal R. S. Free Radic Biol Med 2002; 33:37-44). Most commonlyoccurred oxidative modifications of amino acids include methioninesulfoxidation and tyrosine chlorination, bromination and nitrosylationthat are mediated by such oxidants as H₂O₂, MPO/H₂O₂/Cl⁻, MPO/H₂O₂/Br⁻,and MPO/H₂O₂/NO₂ ⁻, are illustrated in FIG. 4. Oxidative damage tospecific proteins is considered to constitute one of the majormechanisms linking oxidative stress/damage and losses in physiologicalfunctions.

Methionine (Met) is one of the most readily oxidized amino acidconstituents of proteins. It is attacked by H₂O₂, hydroxyl radicals,hypochlorite, chloramines, and peroxynitrite, all these oxidants beingproduced in biological systems. The oxidation product, Met sulfoxide,can be reduced back to Met by peptide methionine sulfoxide reductase(PMSR). Functional changes by Met oxidation in a given protein appear tohave pathophysiological significance in some cases. Formation ofmethionine sulfoxides during oxidative stress has been shown to lead tocomplete inactivation of such physiologically important peptides andproteins as Alzheimer's amyloid beta-peptide 1-42, alpha1-proteaseinhibitor, ribosomal protein L12 and Met-enkephalin (Sohal R. S. FreeRadic Biol Med 2002; 33:37-44; Vogt, W. Free Radic Biol Med 1995;18:93-105). Methionine side chains can be oxidized in vitro usinghydrogen peroxide, which is considered to be a physiological oxidizersince it mimics the methionine oxidation pattern found in vivo.

Those of skill in the art are aware that apolipoprotein (apo) A-Icontains three methionines that can potentially undergo sulfoxidation,Met-86, Met-112, and Met-148 (FIG. 5A). Sulfoxidation of apo A-Imethionines 112 and 148 occurs in vivo (FIG. 5B) and affects many HDLfunctions including uptake by macrophages (von Eckardstein et al. JLipid Res 1991; 32:1465-76; Sigalov et al. Eur J Clin Chem Clin Biochem1997; 35:395-6; Shao et al. Proc Natl Acad Sci USA 2008; 105:12224-9;Shao, B. & Heinecke, J. W. J Lipid Res 2009; 50:599-601; Shao et al.Curr Opin Cardiol 2006; 21:322-8; Shao et al. Curr Opin Mol Ther 2006;8:198-205; Assinger et al. FEBS Lett 2008; 582:778-84; Bergt et al. EurJ Biochem 2001; 268:3523-31; Bergt et al. Arterioscler Thromb Vasc Biol2003; 23:1488-90; Bergt et al. Proc Natl Acad Sci USA 2004; 101:13032-7;Bergt et al. FEBS Lett 1999; 452:295-300). In addition, the content ofoxidized apo A-I between individuals displays the significantvariability (Sigalov A. B. & Stern L. J. FEBS Lett 1998; 433:196-200;von Eckardstein et al. J Lipid Res 1991; 32:1465-76). Apo A-I containingtwo methionine sulfoxides was also observed in human aortic lesions(Pankhurst et al. J Lipid Res 2003; 44:349-55). In summary, thisnaturally occurring apo A-I modification can be suggested to play animportant role in atherogenesis including conversion of native HDL intoa macrophage substrate. In addition, tyrosine chlorination, brominationand nitrosylation (FIGS. 5A-B) have been also reported as a naturallyoccurring oxidative modification of apo A-I (Zheng et al. J Clin Invest2004; 114:529-41; Zheng et al. J Biol Chem 2005; 280:38-47; Panzenboecket al. J Biol Chem 1997; 272:29711-20; Suc et al. J Cell Sci 2003;116:89-99; Pennathur et al. J Biol Chem 2004; 279:42977-83) and tyrosine192 in apo A-I is believed to be the major site of nitration andchlorination by myeloperoxidase in vivo (Shao et al. J Biol Chem 2005;280:5983-93).

In most studies, oxidized HDL generally were either obtained in vitrousing a variety of chemical oxidizing agents under the conditions forwhich oxidation of both lipid and protein HDL constituents can occur orisolated from athrosclerotic tissues but not analyzed for lipidperoxidation products (Bergt et al. FEBS Lett 1999; 452:295-300; Marscheet al. J Biol Chem 2002; 277:32172-9; Panzenboeck et al. J Biol Chem1997; 272:29711-20; Nakano T. & Nagata A. J Lab Clin Med 2003;141:378-84; Hurtado et al. Atherosclerosis 1996; 125:39-46; Nagano etal. Proc Natl Acad Sci USA 1991; 88:6457-61; Nakajima et al. Ann ClinBiochem 2004; 41:309-15; Rifici V. A. & Khachadurian, A. K. BiochimBiophys Acta 1996; 1299:87-94; Pennathur et al. J Biol Chem 2004;279:42977-83; Zheng et al. J Clin Invest 2004; 114:529-41; Zheng et al.J Biol Chem 2005; 280:38-47), thus making impossible to evaluate acontribution of oxidatively damaged protein HDL constituents, ingeneral, and certain oxidative protein modifications, in particular, tothe change of the HDL functional outcome. In addition, the differentoxidants that are widely used to oxidize lipid-free apolipoproteins orapolipoproteins in the context of HDL particles, such as Cu²⁺ ions (Chinet al. J Clin Invest 1992; 89:10-8; Hurtado et al. Atherosclerosis 1996;125:39-46; Nagano et al. Proc Natl Acad Sci USA 1991; 88:6457-61;Nakajima et al. Ann Clin Biochem 2004; 41:309-15), H₂O₂ (Anantharamaiahet al. J Lipid Res 1988; 29:309-18; Sigalov A. B. & Stern L. J. FEBSLett 1998; 433:196-200; Sigalov A. B. & Stern L. J. Chem Phys Lipids2001; 113:133-46; Sigalov A. B. & Stern L. J. Antioxid Redox Signal2002; 4:553-7; von Eckardstein et al. J Lipid Res 1991; 32:1465-76),chloramine T (von Eckardstein et al. J Lipid Res 1991; 32:1465-76),myeloperoxidase-H₂O₂-halide system (Bergt et al. FEBS Lett 1999;452:295-300; Marsche et al. J Biol Chem 2002; 277:32172-9; Panzenboecket al. J Biol Chem 1997; 272:29711-20), cholesteryl ester (Garner et al.J Biol Chem 1998; 273:6088-95; Garner et al. J Biol Chem 1998;273:6080-7) and phosphatidylcholine (Mashima et al. J Lipid Res 1998;39:1133-40) hydroperoxides, and 2,2′-azo-bis(2-amidinopropane)dihydrochloride (AAPH) (Garner et al. J Biol Chem 1998; 273:6080-7;Mashima et al. J Lipid Res 1998; 39:1133-40; Pankhurst et al. J LipidRes 2003; 44:349-55; Panzenbock et al. J Biol Chem 2000; 275:19536-44)modify apo A-I in different ways and to varying extent (see, e.g. FIG.5B). Also, for both myeloperoxidase-H₂O₂-halide system (Bergt et al.Biochem J 2000; 346 Pt 2:345-54) and AAPH (Horkko et al. J Clin Invest1999; 103:117-28), it has been reported that lipid-free and lipid-boundapo A-I is also oxidized in different ways. This variety of theoxidizing reagents used and, therefore, the variety of oxidized HDLstudied has lead to contradictory conclusions about the effect ofoxidative damage to apo A-I on HDL function (Marsche et al. J Biol Chem2002; 277:32172-9; Panzenbock et al. J Biol Chem 2000; 275:19536-44).

Thus, the prior art neither suggests nor teaches one of ordinary skillin the art to investigate the performance of HDL particles in which onlythe apolipoprotein portion has been chemically altered. As describedherein, it is surprisingly found that oxidative modification of onlyprotein constituents or peptide fragments thereof of HDL is sufficientto convert these particles to substrates for macrophage scavengerreceptors.

It is well known to those of ordinary skill in the art that the modifiedapo A-I molecules containing methionine sulfoxides at any one ofpositions 86, 112, 148, or any combination of said positions can beprepared and purified using the standard procedures well known in theart (see e.g. Anantharamaiah et al. J Lipid Res 1988; 29:309-18; SigalovA. B. & Stern L. J. FEBS Lett 1998; 433:196-200; Sigalov A. B. & SternL. J. Chem Phys Lipids 2001; 113:133-46; Sigalov A. B. & Stern L. J.Antioxid Redox Signal 2002; 4:553-7; von Eckardstein et al. J Lipid Res1991; 32:1465-76; Panzenbock et al. J Biol Chem 2000; 275:19536-44). Itshould be also understood by those of ordinary skill in the art that apoA-I peptide fragments containing methionine residues at positions 86,112, 148 can be easily synthesized or manufactured by any technique forpeptide synthesis known in the art, including, e.g., the techniquesdescribed in U.S. Pat. Nos. 6,004,925, 6,037,323 and 6,046,166 and usingthe well known primary sequence of human apo A-I that can be found underthe entry UniProt KB/Swiss-Prot P02647 (www.uniprot.org/uniprot/P02647,last modified on Jul. 28, 2009, version 137). It is further understoodby those of ordinary skill in the art that methionine residues in thesepeptide fragments can be oxidized to methionine sulfoxides using, forexample, the procedures described in (Sigalov A. B. & Stern L. J. FEBSLett 1998; 433:196-200; Sigalov A. B. & Stern L. J. Chem Phys Lipids2001; 113:133-46; Sigalov A. B. & Stern L. J. Antioxid Redox Signal2002; 4:553-7; von Eckardstein et al. J Lipid Res 1991; 32:1465-76;Panzenbock et al. J Biol Chem 2000; 275:19536-44; Garner et al. J BiolChem 1998; 273:6088-95; Garner et al. J Biol Chem 1998; 273:6080-7) andthe modified peptides can be purified by any method known in the art,including high performance liquid chromatography (HPLC).

It is well known to those of ordinary skill in the art that the modifiedapo A-I molecules containing 3-chloro-, 3-nitro- or 3,5-dibromotyrosineat position 192 (FIGS. 4 and 5) can be prepared and purified using thestandard procedures well known in the art (Shao et al. J Biol Chem 2005;280:5983-93; Weiss et al. Science 1986; 234:200-3). It should be alsounderstood by those of ordinary skill in the art that apo A-I peptidefragments containing tyrosine residue at position 192 can be easilysynthesized or manufactured by any technique for peptide synthesis knownin the art, including, e.g., the techniques described in U.S. Pat. Nos.6,004,925, 6,037,323 and 6,046,166 and using the well known primarysequence of human apo A-I that can be found under the entry UniProtKB/Swiss-Prot P02647 (www.uniprot.org/uniprot/P02647, last modified onJul. 28, 2009, version 137). It is further understood by those ofordinary skill in the art that tyrosine residue of these peptidefragments can be oxidized to 3-chloro-, 3-nitro- or 3,5-dibromotyrosine,using, for example, the procedures described in (Shao et al. J Biol Chem2005; 280:5983-93; Weiss et al. Science 1986; 234:200-3) and themodified peptides can be purified by any method known in the art,including high performance liquid chromatography HPLC.

It will be clear to those of ordinary skill in the art that the modifiedapo A-II molecules containing methionine sulfoxide at position 26 can beprepared and purified using the standard procedures well known in theart (see e.g. Anantharamaiah et al. J Lipid Res 1988; 29:309-18; Garneret al. J Biol Chem 1998; 273:6080-7; Sigalov A. B. & Stern L. J. FEBSLett 1998; 433:196-200; Sigalov A. B. & Stern L. J. Chem Phys Lipids2001; 113:133-46; Sigalov A. B. & Stern L. J. Antioxid Redox Signal2002; 4:553-7; von Eckardstein et al. J Lipid Res 1991; 32:1465-76;Panzenbock et al. J Biol Chem 2000; 275:19536-44). It should be alsounderstood by those of ordinary skill in the art that apo A-II peptidefragments containing methionine residue at position 26 can be easilysynthesized or manufactured by any technique for peptide synthesis knownin the art, including, e.g., the techniques described in U.S. Pat. Nos.6,004,925, 6,037,323 and 6,046,166 and using the well known primarysequence of human apo A-II that can be found under the entry UniProtKB/Swiss-Prot P02652 (http://www.uniprot.org/uniprot/P02652, lastmodified on Jul. 28, 2009, version 121). It is further understood bythose of ordinary skill in the art that methionine residue in thesepeptide fragments can be oxidized to methionine sulfoxide using, forexample, the procedures described in (Sigalov A. B. & Stern L. J. FEBSLett 1998; 433:196-200; Sigalov A. B. & Stern L. J. Chem Phys Lipids2001; 113:133-46; Sigalov A. B. & Stern L. J. Antioxid Redox Signal2002; 4:553-7; von Eckardstein et al. J Lipid Res 1991; 32:1465-76;Panzenbock et al. J Biol Chem 2000; 275:19536-44; Anantharamaiah et al.J Lipid Res 1988; 29:309-18) and the modified peptides can be purifiedby any method known in the art, including HPLC.

In preferred embodiments, the chemically or enzymatically modifiedapolipoprotein is selected from a modified apo A-I or a fragment thereofand a modified apo A-II or a fragment thereof. In preferred embodiments,the modified apolipoprotein is any combination of a modified apo A-I anda modified A-II and fragments thereof. In preferred embodiments, amodified apo A-I is an oxidized apoA-I or an oxidized apoA-I fragmentthat comprises one or more of the following amino acid residues:3-chlorotyrosine, 3-nitrotyrosine, 3,5-dibromotyrosine, dityrosine,trihydroxyphenylalanine, dihydroxyphenylalanine, methionine sulphoxide,and tyrosine peroxide. In still other preferred embodiments, a modifiedapo A-II is an oxidized apoA-II or an oxidized apoA-II fragment thatcomprises one or more of the following amino acid residues:chlorotyrosine, nitrotyrosine, dityrosine, trihydroxyphenylalanine,dihydroxyphenylalanine, methionine sulphoxide, and tyrosine peroxide. Inparticularly preferred embodiments, a modified apo A-I is an oxidizedapo A-I or an oxidized apoA-I fragment that comprises methioninesulfoxide at any one of positions 86, 112, 148, or any combination ofsaid positions. In still particularly preferred embodiments, a modifiedapo A-II is an oxidized apo A-II or an oxidized apoA-II fragment thatcomprises methionine sulfoxide at position 26. In still particularlypreferred embodiments, a modified apo A-I is an oxidized apo A-I or anoxidized apoA-I fragment that comprises methionine sulfoxide atpositions 112 and 148.

In certain embodiments, methionine sulfoxidation and the functionalchanges associated with the oxidation can be reversed by PMSR in thepresence of physiologically important universal antioxidantdihydrolipoic acid (DHLA) as a cofactor (Sigalov A. B. & Stern L. J.Antioxid Redox Signal 2002; 4:553-7; Biewenga et al.Arzneimittelforschung 1998; 48:144-8). DHLA is a metabolic productformed in vivo from lipoic acid (Biewenga et al. Gen Pharmacol1997:29:315-31), which is widely used as a therapeutic agent in avariety of diseases. Several lines of evidence suggest that theantioxidant properties of lipoic acid and more importantly, its reducedform, DHLA, are at least in part responsible for the therapeutic effect.Currently, DHLA alone or in equimolar mixture with lipoic acid is widelyused as a supplement. DHLA is also suggested for the amelioration ofdiabetes mellitus types 1 and 2 (including impaired glucose tolerance,pre-diabetes, insulin resistance, metabolic syndrome X and as an adjunctto oral antidiabetic agents and/or insulin), diabetic and non-diabeticmicrovascular diseases (including nephropathy, neuropathy andretinopathy), diabetic and non-diabetic macrovascular diseases(including heart attack, stroke, peripheral vascular disease andischemia-reperfusion injury), hypertension, vasoconstriction, obesity,dyslipedemia, and neurodegenerative disorders (including Parkinson'sdisease, mild cognitive impairment, senile dementia, Alzheimer'sdisease, hearing loss and chronic glaucomas (see e.g., US Pat Appls20090068264, 20020110604, and 20020177558), the disclosures of which areincorporated herein by reference in their entities.

To the extent that the present application is directed to methods andcompositions which employ modified apolipoproteins A-I and A-II andfragments thereof that include protein and peptide molecules containingsulfoxidized methionines, it is important to discuss potential use ofDHLA in combination with one embodiment of the compositions describedherein. This discussion is provided in the section on methods of usingthe rHDL of the present invention.

C. Reconstituted HDL

To the extent that the present application is directed to methods andcompositions which employ HDL, the present section is provided as adiscussion of HDL and major protein constituents of HDL, apolipoproteins(apo) A-I and A-II, that are to be used in the present invention.

Those of skill in the art are aware of methods and compositions forproducing reconstituted lipoproteins (US Pat Appl 20070243136;20060217312; and 20060205643; U.S. Pat. No. 5,652,339; Lerch et al. VoxSang 1996; 71:155-164; Matz C. E. & Jonas, A. J Biol Chem 1982;257:4535-40; Toledo et al. Arch Biochem Biophys 2000; 380:63-70; SigalovA. B. & Stern L. J. Chem Phys Lipids 2001; 113:133-46; Jonas, A. MethodsEnzymol 1986; 128:553-82), in general, and as vehicles for drugdelivery, in particular (Rensen et al. Adv Drug Deliv Rev 2001;47:251-76). As is detailed in Rensen et al., lipoproteins are sphericalmacromolecular particles made up of a hydrophobic core of triglycerides(TG) and cholesteryl esters, which are emulsified by a shell ofamphipathic phospholipids, unesterified cholesterol, and one or moreapolipoproteins. The unesterified sterol and the apolipoproteins in theouter layer of the lipoproteins stabilize the overall structure. Thesenaturally occurring structures circulate in vivo and are intrinsicallyinvolved in lipid transport. These structures are ideal candidates fordrug delivery because being endogenous, these agents are less likely topromote an immune response, and they readily evade detection andelimination through the reticuloendothelial system.

There are four major classes of lipoproteins circulating in human blood,and they differ from each other with respect to size, lipid compositionand apolipoprotein composition. These lipoprotein classes aredistinguishable from each other based on their density inultracentrifugation. All four major classes of circulating lipoproteinparticles are involved in the fat-transport system: chylomicrons, verylow density lipoprotein (VLDL), low density lipoprotein (LDL) and highdensity lipoprotein (HDL). Chylomicrons constitute a short-lived productof intestinal fat absorption. VLDL and particularly, LDL, areresponsible for the delivery of cholesterol from the liver (where it issynthesized or obtained from dietary sources) to extrahepatic tissues,including the arterial walls. HDL, by contrast, mediates reversecholesterol transport (RCT), the removal of cholesterol lipids, inparticular from extrahepatic tissues to the liver, where it is stored,catabolized, eliminated or recycled. HDL also plays a role ininflammation, transporting oxidized lipids and interleukin. Apo A-I, themajor protein constituent of HDL, forms three types of stable complexeswith lipids: small, lipid-poor complexes referred to as pre-beta-1 HDL;flattened discoidal particles comprising polar lipids (phospholipid andcholesterol) referred to as pre-beta-2 HDL; and spherical particles,comprising both polar and nonpolar lipids, referred to as spherical ormature HDL (HDL₃ and HDL₂). Most HDL in the circulating populationcomprise both apo A-I and apo A-II, another protein constituent of HDL,(the “AI/AII-HDL fraction”). However, the fraction of HDL comprisingonly apo A-I (the “AI-HDL fraction”) appears to be more effective inRCT. Certain epidemiologic studies support the hypothesis that the apoA-I/HDL fraction is anti-atherogenic (Parra et al. Arterioscler Thromb1992; 12:701-7; Decossin et al. Eur J Clin Invest 1997; 27:299-307).

In the methods of the present invention, the lipoproteins of interestare synthetic reconstituted HDLs. These lipoproteins are characterizedby a density of about 1.063 g/ml to about 1.21 g/ml, and as such, arethe densest of the four classes of lipoproteins. However, the diameterof these lipoproteins is the smallest of the lipoprotein classes andvaries between 5 to 12 nm. In preferred embodiments of the presentinvention the synthetic rHDL compositions have an average diameter ofbetween about 5 nm and about 15 nm. A particularly preferred range is arange of between 5-10 nm. It is envisioned that in any imagingcomposition of the present invention may contain a range of sizes ofsynthetic rHDL, but the average size of the particles will be in theabove range. Thus, the compositions may have particles that range havediameters of between 5-15 nm, other exemplary ranges of diameters in agiven composition are between 5-10 nm, 5-8 nm, 5-6 nm. In preferredembodiments the average diameter size of the synthetic rHDLs is about 10nm, alternatively, the average diameter is about 6 nm, 7 nm, 8 nm, 9 nm,10 nm, 11 nm, 12 nm, 13 nm, 14 nm or 15 nm. The average size of thesynthetic rHDLs may be as large as 18 nm. Preferably, the above recitedaverage sizes are those sizes that are achieved when the synthetic rHDLhas been reconstituted with at the least the metallic contrast agent.

In those embodiments, in which the imaging composition comprises both ametallic (or non-metallic) agent and an additional targeting agent, itis preferred that the average diameter of the synthetic rHDL moietycomprised of the synthetic rHDL, metallic agent (or non-metallic) andtargeting moiety does not exceed 18 nm.

Schematically, the rHDLs of the present invention that contain modifiedapolipoproteins or fragments thereof as targeting moieties are depictedin FIGS. 1 and 2. In those figures, the metallic contrast agent islinked through a polyaminopolycarboxylate chelating agent that comprisesa hydrophobic group to the phosphate-linked headgroup of a phospholipidmoiety in the rHDL. Of course, it should be understood that this is apreferred embodiment, and those of skill in the art may producesynthetic rHDLs in which the metallic or non-metallic contrast agent iscovalently linked to, or otherwise associated with, another component ofthe synthetic rHDL, such as for example, the apolipoprotein component,or the sterol component. In particular embodiments, it may be desirableto have the metallic or non-metallic contrast agent sequestered in thecore of the synthetic rHDL. For such embodiments, the metallic contrastagent may be linked through a polyaminopolycarboxylate chelating agentto a fatty acyl residue of the TG or even the fatty acyl residue of thecholesteryl ester, or another hydrophobic molecule.

The synthetic rHDL compositions of the present invention in which theparamagnetic metal ion is bonded to a chelator that is attached to thephosphate-linked headgroup of a phospholipid has shown strikingly highcontrast efficiency in MRI imaging, particularly of atheroscleoroticplaques. This is due to the fact that HDL that contain targetingmoieties, the modified apolipoproteins and fragments thereof, are ableto effectively and efficiently locate to such plaques. Such location tothe plaques is further enhanced by the presence of additional targetingmoieties such as e.g., an antibody, or other specific binding partner ofa moiety that is present at the site being imaged.

It is contemplated that the compositions of the present inventionproduce a contrast effect at least 20% better than the contrast seenwhen the metallic ion is presented alone or in phospholipid, micellar,LDL or rHDL (with no apolipoproteins or with unmodified apolipoproteins)forms of delivery. More preferably, the compositions produce a 30%, 35%,40%, 45%, 50%, 55%, 60%, 70% 75% or higher percentage better contrast,than that of comparative compositions of the prior art in which theseother forms of delivery vehicles are used. Without being bound byparticular theory or mechanism, the compositions of the presentinvention are more effective contrast agents than those available in theart because they have the advantage of having a particle sizes between 5and 25 nm, thus being small enough to integrate into the site beingimaged. Further, the agents of the present invention also optionally maycomprise targeting agents that facilitate a increased affinity for theimaged site such that the imaging agent is retained and accumulated atthe site. The preferred agent of the invention preferably has a diameter(if spherical), or longest dimension (if discoidal), of less than 20 nmand greater than 5 nm, more preferably the rHDLs have a diameter orlongest dimension of between about 5 nm and about 15 nm.

As discussed above, the metallic agent preferably comprises a chelatingmoiety that facilitates the conjugation of the metallic agent to forexample, the phospholipid moiety. In certain embodiments, it isdesirable to have the metallic agent chelating moiety present near theouter surface of the synthetic rHDL hydrophobic portion of asphingolipid, and/or the two fatty acyl groups of a phosphatidylcholine,phosphatidylethanolamine, or other glycerophospholipid in the inner sideof the HDL moeity. The fatty acids of the phospholipids may include, butare not limited to, e.g., saturated and unsaturated C₁ to C₂₄ fattyacids. The chelating moiety may be selected from EDTA, DTPA, DTPAGlu,DTPALys, DTPASer, BOPTA, DOTA, DO3A and/or their derivatives, allcontaining a free function unit for covalent linkage to the othermonomer units. The chelating molecule may also be provided with thehydrophobic group in form of a carboxylate amide with hydrophobicaliphatic or aromatic amines. Such amines may be saturated andunsaturated C₁ to C₂₄ amines like methylamine, ethylamine, propylamine,butylamine (n-, iso-, tert-), pentylamine, hexylamine (and isomers),octylamine (and isomers), nonylamine, decylamine, aminoadamantan andfatty amines; as aromatic amines, one may cite substituted andunsubstituted benzyl- and higher phenylalkyl-amines. Alternatively, thepolycarboxylic chelating agent can be provided with lipophilichydrophobic groups linked to the alkylene segments of the molecularback-bone, or to the alpha-carbon of the carboxylate functions or to ahydroxyl group when present in the chelating agent.

In preferred embodiments, the present invention employs Gd-DTPA as themetallic contrast agent comprising the polyaminopolycarboxylate agentchelating agent. In preferred embodiments the chelating agent is DTPA,however, those of skill in the art are aware that other chelating agentscould readily be used in place of DTPA, such other agents include, butare not limited to EDTA, BOPTA, DOTA, DO3A and/or their derivatives. Inthe imaging agents of the invention, the paramagnetic metal may be anyparamagnetic metal traditionally used in MRI techniques and may forexample be selected from e.g., Gd(III), Mn(II), Mn(III), Cr(II),Cr(III), Cu(II), Fe (III), Pr(III), Nd(III) Sm(III), Tb(III), Yb(III)Dy(III), Ho(III), Eu(II), Eu(III), and Er(III), Tl²⁰¹, K⁴², In¹¹¹,Fe.⁵⁹, Tc^(99m), Cr⁵¹, Ga⁶⁷, Ga⁶⁸, Cu⁶⁴, Rb⁸², Mo⁹⁹, Dy¹⁶⁵. In otheraspects of the invention, non-metallic agents are used as contrastagents. Exemplary, but non-limiting non-metallic contrast agents includeFluorescein, Carboxyfluorescein, Calcein, F¹⁸, Xe¹³³, I¹²⁵, I¹³¹, I¹²³,P³², C¹¹, N¹³, O¹⁵, Br⁷⁶, Kr⁸¹. The non-metallic contrast agent maystill preferably be selected from the group of iodinated contrast mediaconsisting of ionic monomers and dimers, and nonionic monomers anddimers, including, but not limiting to, Diatrizoate, Metrizoate,Isopaque, Ioxaglate, Iopamidol, Iohexol, and Iodixanol (Singh J. &Daftary A. J Nucl Med Technol 2008; 36:69-74; Stacul F. Eur Radiol 2001;11:690-7).

A preferred composition for use in the preparation of the rHDLcompositions of the present invention isGd-DTPA-phosphatidylethanolamine (PE). Those of skill in the art areaware of how to produce and use DTPA-PE as a liposomal MRI contrastagent (Grant et al. A liposomal MRI contrast agent:phosphatidylethanolamine-DTPA. Magn Reson Med 1989; 11:236-43). While inpreferred embodiments, it is envisioned that the phospholipid is PE, itshould be understood that the metallic contrast agent may be conjugatedto any phospholipid using techniques such as those that have previouslybeen used to generate DTPA-PE. Such phospholipids include but are notlimited to phosphatidic acid (PA), phosphatidylcholine (PC),phosphatidylethanolamine (PE, particularlydimyristoyl-sn-glycero-phosphatidylethanolamine (DMPE)),phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol(PI), cardiolipin (CL), sphingomyelin (SM), and other sphingolipids. Inaddition to being conjugated to a phospholipid, it may be possible toconjugate the metallic agent to e.g., a mono-phosphate ester of asubstituted or partially substituted glycerol, at least one functionalgroup of said glycerol being esterified by saturated or unsaturatedaliphatic fatty acid, or etherified by saturated or unsaturated alcohol,the other two acidic functions of the phosphoric acid being either freeor salified with alkali or earth-alkali metals. Preferably the phosphateesters will include monophosphates of fatty acid glycerides selectedfrom dimyristoylphosphatidic acid, dipalmitoylphosphatidic acid, ordistearoylphosphatidic acid. Also, it should be understood that thefatty acyl moieties of the phospholipids may vary in length. Forexample, it is envisioned that the phospholipids, TG, cholesterylesters, and monophosphate esters of the glycerol may comprise fattyacids of between C4 to C₂₄ carbons in length. Thus, it should be notedthat that the fatty acyl moieties of the phospholipids may be any fattyacyl moiety commonly found in phospholipids. For example, the fatty acylmoieties may comprise between 4 and 24 carbons and may be saturated, oralternatively may comprise one, two, three or more double bonds.Further, the two fatty acid chains of the phospholipids may be the samefatty acid or alternatively, the phospholipid may comprise two differentfatty acyl moieties. In particularly, preferred embodiments, thephospholipids contain two myristoyl moieties as in DMPE. However, whileDMPE is a preferred phospholipid it is contemplated that DMPC, DMPA,DMPI and the like also may be used.

In preferred embodiments, the phospholipids may also include diacyl anddialkyl glycerophospholipids in which the aliphatic chains have at leasttwelve carbon atoms.

As can be seen from FIGS. 1 and 2, in addition to having phospholipidconjugated to the metallic contrast agent, the rHDLs also comprisephospholipids as part of the overall HDL structure. It is contemplatedthat the rHDL may be made up only of one type of phospholipid or morepreferably, the rHDL will be made up of a mixture of phospholipids.Preferably, in the mixture of phospholipids, the ratio of the individualtypes of phospholipid may be comparable to the ratio of the samephospholipids seen in HDLs circulating in the blood.

In addition to the phospholipid and TG lipid components of the rHDL, thecompositions also comprise sterol esters in the core of the rHDL andsterols interspersed between the phospholipid layer of the rHDL. Whilein preferred embodiments the sterol moiety of the steryl ester and thesterol in the phospholipid layer is cholesterol, it should be understoodthat other common sterols, such as ergosterol, stigmasterol,phytosterol, sitosterol, and lanosterol, also may serve as the sterolmoiety. Other sterols, whether isolated from natural sources orsynthetically generated, also may be used.

The ratios of the various HDL components to each other in the rHDLcompositions of the present invention should be guided by the molar w/wratios well known in the art for HDL moieties (Rensen et al. Adv DrugDeliv Rev 2001; 47:251-76; US Pat Appls 20060217312 and 20060205643;U.S. Pat. No. 5,652,339; Lerch et al. Vox Sang 1996; 71:155-64; Matz C.E. & Jonas A. J Biol Chem 1982; 257:4535-40; Toledo et al. Arch BiochemBiophys 2000; 380:63-70; Sigalov A. B. & Stern L. J. Chem Phys Lipids2001; 113:133-46). In the compositions containing phospholipids, theweight proportion of the phospholipids:steryl ester:sterol:TG may varyin a wide range e.g. from 100±50%:62±50%:25±50%:11±50% with 1-4apolipoprotein molecules per particle. Composition will affect particlesize (because of the surface-to-core ratio), and can even affectparticle shape (discoidal if there is insufficient core lipid). However,those of skill in the art are aware of variations in the ratios ofphospholipids (e.g., DMPC and DPPC) mixed with different ratios of apoA-I (Tall et al. J Biol Chem 1977; 252:4701-11) Compositions such asthose described by Tall et al. may be varied by addition of differingquantities of core lipids as was described, for example, in a laterstudy on phospholipid-enrichment of HDL (Tall A. R. & Green P. H. J BiolChem 1981; 256:2035-44).

In preferred embodiments, the synthetic nanoparticle in the imagingagent comprises a phospholipid:sterol:apolipoprotein ratio of 180:5:3(mol:mol:mol). In other preferred embodiments, the syntheticnanoparticle in the imaging agent comprises aphospholipid:apolipoprotein ratio of 100:3 (mol:mol). In still preferredembodiments, the synthetic nanoparticle in the imaging agent comprises aphospholipid:steryl ester:sterol:triglycerides (TG):apo A-I ratio (w/w)of 100:62:25:11:2. The use of a large excess of chelate may result inunnecessary waste of the chelating/imaging agent while an excess ofphospholipid beyond certain concentration does not provide extrabenefit. Within these ratios, it is contemplated that the ratio ofphospholipid:metallic agent can vary from 75:25 to 0:100, and the ratioof phospholipid:sterol can vary from 5:10 to 20:1. In other embodiments,the rHDL may contain no sterol (i.e., be composed of phospholipids onlywith no sterol).

In preferred embodiments apo A-I_(unox) is unoxidized apo A-I containedin initial serum apo A-I. In other preferred embodiments apo A-II_(unox)is unoxidized apo A-II contained in initial serum apo A-II. In otherpreferred embodiments, apo A-I_(ox) is oxidized apo A-I (withsulfoxidized methionines at positions 112 and 148) contained in serumapo A-I or obtained from unoxidized apo A-I using hydrogen peroxide. Instill other preferred embodiments; apo A-I_(red) is reduced apo A-Iobtained by reduction of oxidized apo A-I (with sulfoxidized methioninesat positions 112 and 148) using peptide methionine sulfoxide reductase(PMSR). In preferred embodiments, rHDL-1 are reconstituted HDL particlescontaining only apo A-I_(unox). In preferred embodiments, rHDL-2 arereconstituted HDL particles containing only apo A-I_(ox). In preferredembodiments, rHDL-3 are reconstituted HDL particles containing apoA-I_(unox) and apo A-I_(ox) with a molar ratio of 1:1. In preferredembodiments, rHDL-4 are reconstituted HDL particles containing apoA-I_(unox), apo A-I_(ox) and apo A-II_(unox), with a molar ratio of3:3:1.

It is contemplated that the compositions of the present invention may beproduced by lyophilisation of the composition whereby a dry, pulverulentformulation is obtained. This form of the paramagnetic composition isparticularly convenient for long term storage. The storage in the powderform is simplified by the fact that reconstitution of the composition beachieved by dispersion of the lyophilised powder in a physiologicallyacceptable liquid carrier, will form a suspension useful as a blood poolNMR imaging contrast agent. The lyophilisation is a straight forwardfreeze-drying process requiring no particular precautions or measures.In certain embodiments, it may be desirable to produce HDLs in alyophilized form and then re-constituted such HDLs using a sonicator oran extruder. Alternatively, the compositions of the present inventioncan by cryopreserved for long storage purposes using standard proceduresof cryopreservation of lipoproteins well known in the art (Rumsey et al.J Lipid Res 1992; 33:1551-61; Sigalov A. B. Eur J Clin Chem Clin Biochem1995; 33:73-81).

The methods for making compositions according to the invention generallycomprise selecting as components a paramagnetic contrast agent with anappropriate polycarboxylic acid chelating agent provided with a suitablelipophilic group in admixture with one or more phospholipids, TGs,sterols and steryl, particularly cholesteryl, esters and apolipoproteinsdispersing the components together so they coalesce into rHDL form.Preferably, the components are dispersed in a physiologically acceptableaqueous liquid carrier such as water or saline, neat or buffered,according to usual practice. Depending upon the choice of components,the dispersion can be achieved by gentle mixing, by detergent dialysis(e.g., cholate dialysis) or by more energetic means such ashomogenization, microfluidization, other shear methods, or sonication.

In specific embodiments, the rHDLs of the present invention that containtargeting moieties of which represent apolipoproteins or fragmentsthereof with sulfoxidized methionine residues may contain dihydrolipoicacid (DHLA) to reverse this oxidative modification at sites of interest.Although it is not necessary to understand the mechanism of aninvention, it is believed that at sites of interest, DHLA serves as acofactor for peptide methionine sulfoxide reductase (PMSR) enzyme toreduce methionine sulfoxides back to their native form.

Those of skill in the art are aware of methods for reconstituting HDLmoieties (Jonas A. Methods Enzymol 1986; 128:553-82). Such methodsinclude detergent mediated synthesis, cosonication of HDL components, orthrough the spontaneous interaction of apolipoproteins with the lipidvesicles and are described below in more detail.

D. Additional Targeting Moieties

As disclosed in US Pat Appl 20070243136, the disclosure of which isincorporated herein by reference, a major advantage of using rHDL isthat while it can freely enter and exit lesions, its steady-state levelsin plaques should be relatively low, unless deliberate steps are made topromote its retention. Ideally, this retention should be dependent onspecific molecules of interest, resulting in an obvious increase insignal intensity (relative to the low background). In the methods andcompositions of the present invention, modified apolipoproteins andfragments thereof serve not only as structural proteins that keepstability, integrity and functionality of rHDL but importantly, asspecific targeting molecules that target rHDL to sites of interest. Inspecific embodiments, modified apolipoproteins and fragments thereof aresubstrates for macrophage scavenger receptors. This causes contrastagent-rHDL particles (i.e., for example, Gd-rHDL particles) to bedelivered and retained in atherosclerotic plaques. Similarly, thiscauses contrast agent-rHDL particles (i.e., for example, Gd-rHDLparticles) to be delivered to tumor-associated macrophages.

It is understood by those of ordinary skill in the art, that alldescribed in the literature methods that enhance binding to and orabsorption by macrophages of lipoprotein nanoparticles can be used inthe present invention. Thus, the imaging agents of the invention mayfurther comprise an additional targeting moiety to further facilitatetargeting of the agent to a specific site in vivo. The additionaltargeting moiety may be any moiety that is conventionally used to targetan agent to a given in vivo site and may include but is not limited to,an antibody, a receptor, a ligand, a peptidomimetic agent, an aptamer, apolysaccharide, a drug and a product of phage display as disclosed in USPat Appl 20070243136 and incorporated herein by reference. In particularembodiments, the targeting moiety may be conjugated to a detectablelabel. For example, apo E-derived lipopeptide (Chen et al. ContrastMedia Mol Imaging 2008; 3:233-42), an apo A-I mimetic peptide (Cormodeet al. Small 2008; 4:1437-44), and murine (MDA2 and E06) or human (IK17)antibodies that bind unique oxidation-specific epitopes (Briley-Saebo etal. Circulation 2008; 117:3206-15) may be used in the present inventionto further improve specific targeting macrophages, decrease the dosageof administered Gd required and increase an image quality of vulnerableplaques.

In preferred embodiments, the targeting moiety is an antibody. By“antibody” the present invention intends to encompass antibody fragmentsand derivatives, thus the term includes, but is not limited to,polyclonal, monoclonal, chimeric, single chain, Fab fragments andfragments produced by a Fab expression library. Such fragments includefragments of whole antibodies which retain their binding activity for anantigen or other marker expressed at the site that is to be targeted bythe rHDL compositions of the invention. Such fragments include Fv,F(ab′) and F(ab′)2 fragments, as well as single chain antibodies (scFv),fusion proteins and other synthetic proteins which comprise theantigen-binding site of the antibody. While the antibodies areprincipally being used herein as targeting agents, such antibodies andfragments thereof may also be neutralizing antibodies, i.e., those whichinhibit biological activity of the polypeptides which they recognize,and therefore may serve the additional purpose of rendering the rHDLcompositions as being useful as diagnostics and therapeutics. Inexemplary embodiments, in the rHDL compositions of the invention Fab orother antibody fragments discussed above can be conjugated to avidinthen complexed to biotinylated PE as described in e.g., Example 2 of USPat Appl 20070243136.

In preferred embodiments, the choice of antibody will be directed byknowledge of plaque biology, which provides a reasonable set ofcandidates for plaques in general and at different stages of developmentas disclosed in US Pat Appl 20070243136 and incorporated herein byreference. For general plaque markers, antibodies against lipoproteinlipase (Williams K. J. & Tabas I. Arterioscler Thromb Vasc Biol 1995;15:551-61; Jonasson et al. J Lipid Res 1987; 28:437-45; Yla-Herttuala etal. Proc Natl Acad Sci USA 1991; 88:10143-7; Babaev et al. J Biol Chem2000; 275:26293-9), oxidized epitopes (O'Brien et al. Circulation 1999;99:2876-82), including oxLDL MDA (Herfst M. J. & van Rees H. ArchDermatol Res 1978; 263:325-334) are suitable. For markers of unstableplaques, antibodies against matrix metalloproteinases (Aikawa et al.Circulation 1998; 97:2433-44) and tissue factor may be used (Rong et al.Circulation 2001; 104:2447-52; Aikawa et al. Circulation 1999;100:1215-22; Badimon et al. Circulation 1999; 99:1780-7; Rauch et al.Ann Intern Med 2001; 134:224-38). The antibodies for these plaquecomponents are either commercially available and known to those of skillin the art and have previously used a number of them in studies of mouseatherosclerosis (e.g., Rong et al. Circulation 2001; 104:2447-52). Also,contemplated for use herein are antibodies or other agents that bindmatrix components. Such agents include apo E and C-reactive protein, aswell as antibodies against biglycan, chondroitin sulfate, and versican(see, for example, Olin-Lewis et al. Circ Res 2002; 90:1333-9). Incertain embodiments, oxidation specific antibodies are used. Those ofskill in the art are referred to Torzewski et al. which provides ateaching in the art of oxidized antibodies to malondialdehyde and usesthereof in imaging plaques and plaque stabilization (Torzewski et al.Arterioscler Thromb Vasc Biol 2004; 24, 2307-12).

For each incorporated antibody, the final rHDL particles should betested as above (size, surface charge, penetration of interstitialfluid, biodistribution and relaxivity) and the results compared tonative and rHDL-Gd-DPTA-PE particles before testing as a plaque imagingagent by undergoing the series of in vivo and ex vivo studies describedbelow.

As disclosed in US Pat Appl 20070243136 and incorporated herein byreference, other markers that can be used as additional targeting agentsinclude arterial-wall sphingomyelinase, a key factor that promoteslipoprotein retention and aggregation (Williams et al. ArteriosclerThromb Vase Biol 1995; 15:551-61; Tabas et al. J Biol Chem 1993;268:20419-32; Marathe et al. Arterioscler Thromb Vase Biol 1999;19:2648-58; Schissel et al. J Biol Chem 1998; 273:2738-46; Schissel etal. J Clin Invest 1996; 98:1455-64; Williams et al. Curr Opin Lipidol1998; 9:471-4). Sphingomyelinase is abundant in atherosclerotic plaques,and its role in atherogenesis is to digest sphingomyelin inlipoproteins, thereby generating ceramide. Ceramide is a fusogen thatcauses the lipoproteins to aggregate, forming large complexes that canno longer leave the plaque. There are two conditions required forsphingomyelinase to mediate this process: i) the lipoproteins must havea sufficiently high content of sphingomyelin, to allow efficientdigestion by arterial-wall sphingomyelinase (Schissel et al. J Biol Chem1998; 273:2738-46); and ii) the particles must initially remain in thelesion long enough to become digested. Thus, certain of the rHDLcompositions of the invention comprise both an antibody to promote someinitial retention, but also a high sphingomyelin content to promotedigestion by sphingomyelinase to increase particle aggregation andtrapping in the plaque. Thus, based on a well-characterizedpathophysiologic process, sphingomyelin containing rHDL compositions ofthe invention will amplify the signal from antibody-mediated retentionof Gd-rHDL in atherosclerotic plaques. In addition, it is contemplatedthat the agents of the invention may be targeted to the extracellularmatrix components. Illustrative, but not restrictive, examples ofextracellular matrix components include, but are not limited to, aproteoglycan, a chondroitin sulfate proteoglycan, a heparan sulfateproteoglycan, a mixed proteoglycan, versican, perlecan, biglycan,decorin, a small leucine-rich proteoglycan, a syndecan, a glypican,betaglycan, macrophage colony-stimulating factor, a collagen, a type Icollagen, a type III collagen, an elastin, a fibronectin, a laminin, anon-proteoglycan, a macrophage-derived molecule, a smooth musclecell-derived molecule, a mast cell-derived molecule, a molecule derivedfrom an inflammatory cell, a molecule derived from a non-inflammatorycell, an endothelial-derived molecule, and a cell-derived matrixcomponent.

Further it has been shown that the apoAI moiety of HDL interacts withABCA1 and undergoes an intracellular travel route (calledretroendocytosis; Bared et al. Mol Biol Cell 2004; 15:5399-407). It iscontemplated that the proteins encountered during this route would be inclose proximity to HDL. The rHDL compositions of the present inventioncontain at least one modified apolipoprotein molecule per particlewhereas other apolipoprotein moieties can be unmodified. As such, therHDL compositions of the present invention, with or without additionaltargeting agents, could be employed to transport materials tointracellular locations within the cell. It is contemplated thatmacrophage intracellular localization of the Gd can be achieved usingthe compositions of the present invention, consistent with theretroendocytosis pathway delivery HDL to the interior of the cells.Since HDL interacts with ABCAI, by extension, the HDL may furtherinteract with proteins associated with ABCA1, the rHDL compositions ofthe invention can those be used to target such intracellular proteins.Other transporters in the ABC family, such as ABCG1 and ABCG4, andSR-BI, all known to interact with HDL, may also serve as “bridges”between the rHDL with imaging (with or without additional targetingagents) so that the presence of a variety of intracellular molecules maybe sensed. Besides assessing the presence of such molecules, therHDL-imaging agent approach may also be useful to explore cellularpathways—i.e., the subcellular localization and transfers of the rHDLwould serve as a tracer and could provide direct physical evidence ofany process used in the cellular itinerary of rHDL. Proteins thatinteract with ABCA1 or ABC-family proteins include CFTR, GTPases(Cdc42), apoptosis proteins (FADD, PDZ proteins (Beta2-syntrophin, alpha1-syntrophin, Lin7), SNARE proteins (syntaxin-13, syntaxin 1A, SNAP-23),phagosome proteins (flotillin-1, syntaxin-13). The rHDL compositionscould be used to target the plasma membrane or intracellular locationswithin endosomes and lysosomes.

As disclosed in US Pat Appl 20070243136 and incorporated herein byreference, cell surface proteins that may serve as an additional targetfor the rHDL compositions of the invention can be divided up into 3groups-receptors, adhesion molecules, and miscellaneous proteins. Theproteins should be on the cell surface of the major types of cells foundin plaques; i.e., endothelial cells, macrophages, smooth muscle cells,and lymphocytes.

Receptors that can serve as main and additional targets in the presentinvention include, e.g., scavenger receptor, SR-BI, LDLR, LRP, apo Ereceptor, VLDL receptor, CD36, oxidized LDL receptor, sphingosine-1Preceptor, CD44. Of course there are many other receptors, such as FGF,insulin, EGF, etc, that may also be targeted. Adhesion molecules thatmay be targeted include e.g., VCAM, ICAM, cadherins, integrins,selectins, and their binding partners (which are also cell surfacemolecules). In specific embodiments, it is contemplated that the skilledartisan could prepare rHDL compositions to additionally target T cellsfor imaging with reagents against CD2 (all T cells) CD3 (all T cells),CD4 (major subset of T cells), CD8 (major subset of T cells), and CD90(all T cells). Markers for B cells include, e.g., B220 (a Bcell-specific isoform of CD45), CD19 and CD20 (both useful pan-targetsfor both human and mouse B cells). Other useful markers may includeNK1.1 that targets NKT cells as well as CD3 (for T cells).

Of course, in addition to antibodies other agents may be used asadditional targeting moieties. Such agents include, but are not limitedto, ligands for receptors (or vice versa) that are expressed on thesurface of a given site to be targeted.

In certain embodiments, the additional targeting moiety is an aptamer.Aptamers are DNA or RNA molecules that have been selected from randompools based on their ability to bind other molecules. Aptamers have beenselected which bind nucleic acid, proteins, small organic compounds, andeven entire organisms. Methods and compositions for identifying andmaking aptamers are known to those of skill in the art and are describede.g., in U.S. Pat. Nos. 5,840,867 and 5,582,981 each incorporated hereinby reference. In addition to aptamers, DNA, RNA, or modified DNA or RNAmolecules also may be included as targeting moieties.

Receptors such as e.g., cytokine receptors may specifically be targetedby using a cytokine having a receptor binding domain capable ofinteracting with a cell receptor site. Such cytokines include but arenot limited to IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12, IL-13, EPO, G-CSF, M-CSF, GM-CSF, IGF-1, and LIF.It should be understood that these are merely exemplary ligands ofcertain receptors and the compositions of the invention may readily beadapted to comprise any ligand to any receptor that is expressed on thecell surface, so long as that ligand can be modified to be attached toe.g., a phospholipid moiety of the rHDLs of the present invention andyet retain its receptor binding capability. In addition, C-reactiveprotein (CRP) and/or to activated complement could also be targeted.Those of skill in the art are referred to Torzewski et al., (Torzewskiet al. Arterioscler Thromb Vasc Biol 1998; 18:1386-92) and which provideadditional teachings of the role of CRP in the arterial intima and showthat CRP frequently colocalizes with the terminal complement complex inthe intima of early atherosclerotic lesions of human coronary arteries.As such, targeting CRP will likely be useful in imaging atheroscleroticlesions at an early stage of atherosclerosis. Drugs designed to treatatherosclerosis may be added to the gadolinium-based complexes toachieve an effective intervention of the disorder at an early stage.Example is DHLA (or its precursor, lipoic acid, LA) which is well knownto serve as a cofactor for PMSR and reduce sulfoxidized P oa hR nf 111methionines back to their native form (Biewenga et al.Arzneimittelforschung 1998; 48:144-8; Sigalov A. B. & Stern L. J.Antioxid Redox Signal 2002; 4:553-7)

E. Methods of Making Reconstituted Lipoprotein Complexes

As disclosed in US Pat Appl 20070243136 and incorporated herein byreference, the lipoprotein complexes described herein can be prepared ina variety of forms, including, but not limited to vesicles, liposomes,proteoliposomes, micelles, discoidal and spherical particles. A varietyof methods well known to those skilled in the art can be used to preparethe charged lipoprotein complexes. A number of available techniques forpreparing liposomes or proteoliposomes may be used. For example,apolipoprotein can be co-sonicated (using a bath or probe sonicator)with the appropriate phospholipids to form complexes. Alternatively,apolipoprotein can be combined with preformed lipid vesicles resultingin the spontaneous formation of charged lipoprotein complexes (U.S. Pat.No. 6,248,353). The charged lipoprotein complexes and liposomal HDL-likecompositions can also be formed by a detergent dialysis method; e.g., amixture of apolipoprotein, charged phospholipid(s) SM and/or lecithinand a detergent such as cholate is dialyzed to remove the detergent andreconstituted to form lipoprotein complexes, or by using an extruderdevice or by homogenization (see, e.g., Jonas A. Methods Enzymol 1986;128:553-82; US Pat Appls 20090311191 and 20100202974).

In some embodiments, charged lipoprotein complexes can be prepared bythe cholate dispersion method described in US Pat Appls 20040067873 and20060217312, the disclosures of which is incorporated herein byreference. Cholate can be removed by methods well known in the art. Forexample cholate can be removed by dialysis, ultrafiltration or byremoval of cholate molecules by adsorption absorption onto an affinitybead or resin. In one embodiment, the affinity beads, e.g., BIO-BEADS®(Bio-Rad Laboratories) are added to the preparation of chargedlipoprotein complexes and cholate to adsorb the cholate. In anotherembodiment, the preparation, e.g., a micellar preparation of the chargedlipoprotein complexes and cholate, is passed over a column packed withaffinity beads.

In a specific embodiment, cholate is removed from a preparation ofcharged lipoprotein complexes by loading the preparation onto BIO-BEADS®within a syringe. The syringe is then sealed with barrier film andincubated with rocking at 4° C. overnight. Before use, the cholate isremoved by injecting the solution through BIO-BEADS®, where it isadsorbed by the beads.

In preferred embodiments, the rHDL complexes are prepared by the sodiumcholate dialysis method essentially as described (Sorci-Thomas et al. JBiol Chem 1998; 273:11776-82) with an initial molar ratio of sodiumcholate-POPC-cholesterol-apo A-I of 150:80:4:1. This method has beenused previously to prepare rHDL with 2 or 3 apo A-I per particle and9-11 nm diameter (Sorci-Thomas et al. J Biol Chem 1998; 273:11776-82;Durbin D. M. & Jonas A. J Biol Chem 1997; 272:31333-9; Toledo et al.Arch Biochem Biophys 2000; 380, 63-70; Davidson et al. J Biol Chem 1995;270:5882-90; Sigalov A. B. & Stem, L. J. Chem Phys Lipids 2001;113:133-46).

Stable preparations having a long shelf life may be made bylyophilization. For example, the co-lyophilization procedure describedbelow provides a stable formulation and ease of formulation/particlepreparation process. Co-lyophilization methods are also described inU.S. Pat. No. 6,287,590, which is incorporated herein by reference inits entirety. The lyophilized charged lipoprotein complexes can be usedto prepare bulk supplies for pharmaceutical reformulation, or to prepareindividual aliquots or dosage units that can be reconstituted byrehydration with sterile water or an appropriate buffered solution priorto administration to a subject.

U.S. Pat. Nos. 6,004,925, 6,008,202, 6,037,323, 6,046,166, 6,287,590,7,588,751, and US Pat Appl 20090312402 (incorporated herein by referencein their entireties) disclose a simple method for preparing chargedlipoprotein and liposomal complexes that have characteristics similar toHDL. This method, which involves co-lyophilization of apolipoprotein andlipid solutions in organic solvent (or solvent mixtures) and formationof charged lipoprotein complexes during hydration of the lyophilizedpowder, has the following advantages: (1) the method requires very fewsteps; (2) the method uses inexpensive solvent(s); (3) most or all ofthe included ingredients are used to form the designed complexes, thusavoiding waste of starting material that is common to the other methods;(4) lyophilized complexes are formed that are very stable during storagesuch that the resulting complexes may be reconstituted immediatelybefore use; (5) the resulting complexes usually need not be furtherpurified after formation and before use; (6) toxic compounds, includingdetergents such as cholate, are avoided; and (7) the production methodcan be easily scaled up and is suitable for GMP manufacture (i.e., in anendotoxin-free environment).

In some embodiments, co-lyophilization methods commonly known in the artare used to prepare charged lipoprotein complexes. Briefly, theco-lyophilization steps include solubilizing the apolipoprotein andphospholipids together in an organic solvent or solvent mixture, orsolubilizing the apolipoprotein and phospholipids separately and mixingthem together. The desirable characteristics of solvent or solventmixture are: (i) a medium relative polarity to be able to dissolvehydrophobic lipids and amphipatic protein, (ii) solvents should be class2 or 3 solvent according to FDA solvent guidelines (Federal Register,volume 62, No. 247) to avoid potential toxicity associated with theresidual organic solvent, (iii) low boiling point to assure ease ofsolvent removal during lyophilization, (iv) high melting point toprovide for faster freezing, higher temperatures of condenser and, henceless ware of freeze-dryer. In a preferred embodiment, glacial aceticacid is used. Combinations of e.g., methanol, glacial acetic acid,xylene, or cyclohexane may also be used.

The apolipoprotein/lipid solution is then lyophilized to obtainhomogeneous apolipoprotein/lipid powder. The lyophilization conditionscan be optimized to obtain fast evaporation of solvent with minimalamount of residual solvent in the lyophilized apolipoprotein/lipidpowder. The selection of freeze-drying conditions can be determined bythe skilled artisan, and depends on the nature or solvent, type anddimensions of the receptacle, e.g., vial, holding solution, fill volume,and characteristics of freeze-dryer used. The concentration oflipid/apolipoprotein solution prior to the lyophilization, for organicsolvent removal and successful formation of complexes, can range from 10to 50 mg/ml concentration of apo A-I equivalent and from 20 to 100 mg/mlconcentrations of lipid.

The apolipoprotein-lipid complexes form spontaneously after hydration ofapolipoprotein-lipid lyophilized powder with an aqueous media ofappropriate pH and osmolality. In some embodiments, the media may alsocontain stabilizers such as sucrose, trehalose, glycerin and others. Insome embodiments, the solution must be heated several times abovetransition temperature for lipids for complexes to form. The molar ratioof lipid to protein for successful formation of charged lipoproteincomplexes can be from 2:1 to 200:1 (expressed in apoA-I equivalents) andis preferably about 2:1 weight of lipid to weight of protein (wt/wt).Powder is hydrated to obtain final complex concentration of about 5-30mg/ml expressed, in apoA-I protein equivalents.

In some embodiments, apolipoprotein powder is obtained by freeze-dryingapolipoprotein solution in NH₄CO₃ aqueous solution. A homogeneoussolution of apolipoprotein and lipids is formed by dissolving theirpowders and apolipoprotein in glacial acetic acid. The solution is thenlyophilized, and HDL-like charged lipoprotein complexes are formed byhydration of lyophilized powder with aqueous media.

In some embodiments, homogenization is used to prepareapolipoprotein-lipid complexes. This method may be used to prepareapolipoprotein/soybean-PC complexes and is routinely used forformulation of apoA-I_(Milano)-POPC complexes. Homogenization can beeasily adapted for formation of charged lipoprotein complexes. Briefly,this method comprises forming a suspension of lipids in aqueous solutionof apolipoprotein by Ultraturex™, and homogenization of formedlipid-protein suspension using high-pressure homogenizer untilsuspension becomes clear-opalescent solution and complexes are formed.Elevated temperatures above lipid transition are used duringhomogenization. Solution is homogenized for extended period of time 1-14hours and elevated pressure.

In some embodiments, lipoprotein complexes can be formed byco-lyophilization of phospholipid with peptide or protein solutions orsuspensions. The homogeneous solution of peptide/protein, phospholipids,SM and/or lecithin (plus any other phospholipid of choice) in an organicsolvent or organic solvent mixture can be lyophilized, and chargedlipoprotein complexes can be formed spontaneously by hydration of thelyophilized powder with an aqueous buffer. Examples of organic solventsor their mixtures are include, but are not limited to, acetic acid,acetic acid and xylene, acetic acid and cyclohexane, and methanol andxylene.

A suitable proportion of protein (peptide) to lipid can be determinedempirically so that the resulting complexes possess the appropriatephysical and chemical properties; i.e., usually (but not necessarily)similar in size to HDL. The resulting mixture of apolipoprotein andlipid in solvent is frozen and lyophilized to dryness. Sometimes anadditional solvent must be added to the mixture to facilitatelyophilization. It is expected that this lyophilized product will beable to be stored for long periods and will remain stable.

The lyophilized product can be reconstituted in order to obtain asolution or suspension of the charged lipoprotein complex. To this end,the lyophilized powder is rehydrated with an aqueous solution to asuitable volume (typically 5-20 mg charged lipoprotein complex/ml) whichis convenient for e.g., intravenous injection. In a preferred embodimentthe lyophilized powder is rehydrated with phosphate buffered saline,saline bicarbonate, or a physiological saline solution. The mixture maybe agitated or vortexed to facilitate rehydration. In general, thereconstitution step should be conducted at a temperature equal to orgreater than the phase transition temperature of the lipid component ofthe complexes. Within minutes of reconstitution, a clear preparation ofreconstituted charged lipoprotein complexes should result.

Other methods include spray-drying, where solutions are sprayed andsolvent evaporated (either at elevated temperatures or at reducedpressure). Lipids and apolipoproteins could be solubilized in the samesolvent or in different solvents. Powder filling can then be used tofill vials.

Lyophilized powder from apolipoproteins and lipids could also be mixedmechanically. Homogeneous powder containing the apoplipoprotein andlipids could then be hydrated to form spontaneously complexes of theappropriate size and the appropriate lipid:apolipoprotein molar ratio.

An aliquot of the resulting reconstituted preparation can becharacterized to confirm that the complexes in the preparation have thedesired size distribution; e.g., the size distribution of HDL.Characterization of the reconstituted preparation can be performed usingany method known in the art, including, but not limited to, sizeexclusion filtration, gel filtration, column filtration, gel permeationchromatography, and non-denaturating gel electrophoresis.

For example, after hydration of lyophilized charged lipoprotein powderor at the end of homogenization or cholate dialysis formedapolipoprotein/lipid HDL-like particles are characterized with respectto their size, concentration, final pH and osmolality of resultingsolution, in some instances, integrity of lipid and/or apolipoproteinare characterized. The size of the resulting charged lipoproteinparticles is determinative of their efficacy, therefore this measurementis typically included for characterization of the particles.

In some embodiments, gel permeation chromatography (GPC), e.g., a highpressure liquid chromatography system equipped with a 1×30 cm Superdex™column (Pharmacia Biotech) and UV-detector may be used. Complexes areeluted with bicarbonate buffered saline comprised of 140 mM NaCl and 20mM sodium bicarbonate delivered with 0.5 ml/min flow rate. A typicalamount of complex injected is 0.1 to 1 mg based on protein weight. Thecomplexes can be monitored by absorbance at 280 nm.

Protein and lipid concentration of charged lipoprotein particlessolution can be measured by any method known in the art, including, butnot limited to, protein and phospholipid assays as well as bychromatographic methods such as HPLC, gel filtration chromatography, GCcoupled with various detectors including mass spectrometry, UV ordiode-assay, fluorescent, elastic light scattering and others. Theintegrity of lipid and proteins can be also determined by the samechromatographic techniques as well as peptide mapping, SDS-PAGE, N- andC-terminal sequencing for proteins and standard assays to determinelipid oxidation for lipids.

The homogeneity and/or stability of the lipoprotein complexes orcomposition described herein can be measured by any method known in theart, including, but not limited to, chromatographic methods such as gelfiltration chromatography. For example, in some embodiments a singlepeak or a limited number of peaks can be associated with a stablecomplex. The stability of the complexes can be determined by monitoringthe appearance of new of peaks over time. The appearance of new peaks isa sign of reorganization among the complexes due to the instability ofthe particles.

The optimum ratio of phospholipids to apolipoprotein(s) in the chargedcomplexes can be determined using any number of functional assays knownin the art, including, but not limited to, gel electrophoresis mobilityassay, size exclusion chromatography, interaction with HDL receptors,recognition by ATP-binding cassette transporter (ABCA1), uptake by theliver, binding to and uptake by macrophages, andpharmacokinetics/pharmacodynamics. For example, gel electrophoresismobility assays can be used to determine the optimum ratio ofphospholipids to apolipoproteins in the charged complexes. The chargedcomplexes described herein should exhibit an electrophoretic mobilitythat is similar to natural pre-beta-HDL or alpha-HDL particles. Thus, insome embodiments, natural pre-beta-HDL or alpha-HDL particles can beused as standard for determining the mobility of the charged complexes.

As another example, size exclusion chromatography can be used todetermine the size of the charged complexes described herein as comparedto natural pre-beta-HDL particles. Natural pre-beta-HDL particlesgenerally are not larger than 10-12 nm, and discoidal particles areusually around 7-10 nm.

As another example, HDL receptors can be used in a functional assay toidentify which complex is closest to natural pre-beta-HDL particles, orto identify which complex is the most effective in removing and/ormobilizing cholesterol or lipids from a cell. In one assay, thecomplexes can be tested for their ability to bind ABCA-1 receptors. Suchan assay can differentiate ABCA-1 dependent on independent removal ofcholesterol. Even though apoA-I is considered the best ligands for suchan assay, complexes such as small micellar or small discoidal particlesare also potent ABCA-I ligands. ABCA-1 binding assays that can be usedare described (Brewer et al. Arterioscler Thromb Vasc Biol 2004; 24,1755-60).

As another example, ABCA1 expressing cells are known to recognize freeapoA-1 and to a lesser extent, natural pre-beta-HDL particles (Brewer etal. Arterioscler Thromb Vasc Biol 2004; 24, 1755-60). In theseembodiments, recognition of ABCA1 cells of natural pre-beta-HDLparticles can be compared to any one of the complexes described hereinto identify the complex that most closely resembles natural pre-beta-HDLparticles.

As another example, macrophage scavenger receptors can be used in afunctional assay to identify which complex is the most effective inbeing absorbed and/or uptaken by macrophages. In one assay, thecomplexes can be tested for their ability to bind macrophage scavengerreceptors. Such an assay can differentiate macrophage scavengerreceptor-dependent on independent binding to and/or uptake bymacrophages. Assays to assess binding, uptake and degradation of thecompositions of the present invention by macrophages that can be usedherein are well known in the art (e.g., Suc et al. J Cell Sci 2003;116:89-99; Panzenboeck et al. J Biol Chem 1997; 272:29711-20; Musanti R.& Ghiselli G. Arterioscler Thromb 1993; 13:1334-45; Thorne et al. FEBSLett 2007; 581:1227-32).

A relatively simple approach for identifying lipoprotein complexes thatmost closely resemble natural pre-beta-HDL particles is to perfuselivers with a solution containing the reconstituted charged complexesand measure the amount that is taken up by the liver.

In some embodiments, the pharmacokinetics/pharmacodynamics (PK/PD) ofthe compositions of the invention can be measured following a singleinjection in rabbits. In these embodiments, the concentration of apo A-1or apo A-II (or fragments thereof) is used as a marker of the kinetics.The pharmacodynamics can be measured as the amount of cholesterolmobilized above baseline after a single injection, as well s the amountof cholesterol in the HDL fraction. PK and PD depend on the nature ofthe phospholipids, the composition of the phospholipids, thelipid:apolipoprotein molar ratio and the phospholipid concentration ofthe complex. For example, dipalmitoylphosphatidylcholine (DPPC)/apoA-1complexes have a longer half-live than egg phosphatidylcholine(EPC)/apoA-I complexes. Sphingomyelin/apoA-1 complexes have a longerhalf-life than EPC/apoA-1 complexes. The half-life of human apoA-1 inhumans is approximately 5 to 6 days.

F. rHDL Moieties as Drug Delivery Vehicles

As disclosed in US Pat Appl 20070243136 and incorporated herein byreference, in certain embodiments, it may be desirable to have rHDLmoieties in which the metallic contrast agent is conjugated to acomponent of the core of the HDL, such as e.g., a cholesteryl ester.Methods and compositions for making DTPA mono- and di-stearyl esterscorresponding to metal, e.g., gadolinium chelates, or non-metals, e.g.,iodine, are known to those of skill in the art and have previously beendescribed (Kabalka et al. Magn Reson Med 1988; 8:89-95; Torchilin V. P.Mol Med Today 1996; 2:242-9; Weissig et al. Colloids Surf BBiointerfaces 2000; 18:293-9) Such methods may be used to produce rHDLsin which the rHDLs of the present invention comprise a targetingapolipoprotein moiety at the surface and a metallic or non-metallicagent in the core. Such compositions may be useful, for example, totarget the imaging agent to a specific site and to later apply an enzymeor other composition that would promote the metabolism or breakdown ofthe HDL particle, such as a lipoprotein lipase, cholesteryl estertransfer protein or a phospholipid transfer protein, to effect releasethe imaging agent in a control manner. Imaging agent can be releasedenzymatically. The characterization of the signal can change and be usedto improve detection of any disease/disorder or any organ in the body.Such a composition would be useful is the imaging agent was being usedto track the delivery of a drug or other therapeutic component to aspecific in vivo site.

Therefore, it is contemplated that the compositions of the presentinvention, in addition to comprising a targeting moiety and a metallicor non-metallic contrast agent, also may comprise a third agent that isbeing delivered to effect a therapeutic outcome. Any agent can bedelivered in this manner and methods of using lipoproteins to deliverdrugs are well known to those of skill in the art (Rensen et al. AdvDrug Deliv Rev 2001; 47:251-276). The therapeutic agent that may be usedin the compositions of the invention is limited only by the featuresthat it should not destroy the structural integrity of the rHDL particleor render it larger than 18 nm. Further, the drug should be such that itdoes not quench or otherwise interfere with the signal generated by themetallic or non-metallic ion.

In specific embodiments, the compositions of the present invention maycontain naturally occurring DHLA or its precursor, lipoic acid, LA, todeliver these therapeutic agents to sites of interest such, for example,as atherosclerotic plague. DHLA is a metabolic product formed in vivofrom lipoic acid (Biewenga et al. Gen Pharmacol 1997; 29:315-31), whichis widely used as a therapeutic agent in a variety of diseases. Severallines of evidence suggest that the antioxidant properties of lipoic acidand more importantly, its reduced form, DHLA, are at least in partresponsible for the therapeutic effect. Currently, DHLA alone or inequimolar mixture with lipoic acid is widely used as a supplement. DHLAis also suggested for the amelioration of diabetes mellitus types 1 and2 (including impaired glucose tolerance, pre-diabetes, insulinresistance, metabolic syndrome X and as an adjunct to oral antidiabeticagents and/or insulin), diabetic and non-diabetic microvascular diseases(including nephropathy, neuropathy and retinopathy), diabetic andnon-diabetic macrovascular diseases (including heart attack, stroke,peripheral vascular disease and ischemia-reperfusion injury),hypertension, vasoconstriction, obesity, dyslipedemia, andneurodegenerative disorders (including Parkinson's disease, mildcognitive impairment, senile dementia, Alzheimer's disease, hearing lossand chronic glaucomas (see e.g., US Pat Appls 20090068264, 20020110604,and 20020177558), the disclosures of which are incorporated herein byreference in their entities. In addition, DHLA is well known in the artto serve as a cofactor for PMSR and reduce sulfoxidized methionines backto their native form (Biewenga et al. Arzneimittelforschung 1998;48:144-8; Sigalov A. B. & Stern L. J. Antioxid Redox Signal 2002;4:553-7). Therefore, it is contemplated, the compositions of the presentinvention, in addition to comprising a targeting moiety and a metallicor non-metallic contrast agent, also may comprise DHLA and/or LA that isbeing delivered to effect a therapeutic outcome (for example, to reducemethionine sulfoxides in human apolipoproteins A-I and A-II ofatherosclerotic plagues back to their native form). In certainembodiments, the modified apolipoprotein targeting moieties of the rHDLcompositions of the invention are methionine sulfoxide-containingapolipoproteins A-I, A-II and/or fragments thereof. Incorporation ofDHLA and/or LA in these compositions may result in reducingapolipoprotein methionine sulfoxides of the rHDL compositions back tonative methionines after delivery of contrast agents to sites ofinterest such, for example, as atherosclerotic plagues.

G. Methods of Using the rHDL

The rHDL and HDL-like liposomal compositions of the present inventionwill be useful in any imaging techniques such as computed tomography(CT), gamma-scintigraphy, positron emission tomography (PET), singlephoton emission computed tomography (SPECT), magnetic resonance imaging(MRI), and combined imaging techniques. These compositions will provideeffective delivery of a contrast agent to macrophage-rich sites ofinterest in vivo. In particularly preferred embodiments, thecompositions are used to image atherosclerotic plaques. As disclosed inUS Pat Appl 20070243136 and incorporated herein by reference, suchtechniques have previously been used in pigs, primates and humans aswell as mice and other animals to document, e.g., progression andregression of atherosclerosis in vivo (Skinner et al. Nat Med 1995;1:69-73; McConnell et al. Arterioscler Thromb Vasc Biol 1999; 19;1956-9; Worthley et al. Circulation 2000; 101:2956-61; Worthley et al.Circulation 2000; 101:586-9; Johnstone et al. Arterioscler Thromb VascBiol 2001; 21:1556-60; Helft et al. Circulation 2002; 105:993-8; Helftet al. J Am Coll Cardiol 2001; 37:1149-54; Li et al. Radiology 2001;218:670-8; Lin et al. J Magn Reson Imaging 1997; 7:183-90; Corti et al.J Am Coll Cardiol 2002; 39:1366-1373; Toussaint et al. Circulation 1996;94:932-8; Yuan et al. Circulation 1998; 98:2666-71; Coulden et al. Heart2000; 83:188-91; Hatsukami et al. Circulation 2000; 102:959-64; Fayad etal. Ann N Y Acad Sci 2000; 902:173-86; Fayad et al. Circulation 2000;101:2503-9; Botnar et al. Circulation 2000; 102:2582-7; Jaffer et al.Arterioscler Thromb Vasc Biol 2009; 29:1017-24; Ouhlous et al. J MagnReson Imaging 2002; 15:344-51; Cai et al. Circulation 2002; 106:1368-73;Weissleder R. Nat Rev Cancer 2002; 2, 11-8). Any such techniques may nowbe modified and conducted using the compositions of the presentinvention.

In addition, as disclosed in US Pat Appl 20070243136 and incorporatedherein by reference, it is contemplated that, as HDLs are foundcirculating within the blood, the compositions of the invention may beused in imaging other tissues and sites within the body. As HDLs havethe advantage of not being atherogenic, these compositions may be usedin blood pool analyses to facilitate imaging of, e.g., myocardial andcerebral ischemia, pulmonary embolism, vascularization of tumors, tumorimaging, tumor perfusion, and the like. Given that the rHDL compositionsof the invention comprise targeting agents, rHDLs may be designed totarget any site within the body that contains a site-specific markersuch, for example, as macrophages in atherosclerotic plagues. As HDL isable to enter into interstitial fluid in general (i.e., across theendothelial layer of all blood vessels), the rHDL of the invention maybe used to deliver imaging agents and drugs to any site.

The compositions will generally be injected into the animal to beimaged. By way of example, and in order to test the efficacy of the rHDLcompositions, the rHDL compositions may be injected into mice or othertest animals and their targeting to a site of interest may thus bedetermined. Preferably, prior to injection into mice, the rHDL will betested for pyrogens. The initial volume to be injected is not expectedto exceed about 0.3 ml per mouse, based on the projected rHDL dose ofabout 8 mg of apo A-I or equivalent dose of apo A-II and/or fragmentsthereof per mouse and previous literature indicating that rHDL mixturescontaining about 26 mg apoA-I/ml can be prepared and injected withoutdifficulty (Shah et al. Circulation 2001; 103:3047-50). This volume canbe routinely injected by jugular vein. This route has the addedadvantage of allowing an increase in the volume of injectate, in case itbecomes necessary to increase the amount of Gd to increase the signalbeing generated.

Those of skill in the art also routinely monitor in vitro relaxivitymeasurement for contrast agents. It is contemplated that the contrastagents in the imaging compositions of the present invention have agreater relaxivity than the metallic contrast agents being administeredthrough conventional methods because of the substantial load of Gd onrHDL. Any increase in relativity of the instant contrast agents ascompared related compositions prepared in other non-HDL vehicles will bean advantageous property of the compositions of the present invention.Preferably, there is a 30-250% greater relaxivity of the presentcompositions as compared to those known to those of skill in the art.Thus, the higher relaxivities coupled to the specific targeting of thecontrast agent using the rHDL compositions of the invention provide animportant advance (advantage) in comparison to the known in the art NMRcontrast agent compositions.

In specific embodiments, in vitro relaxivity measurements ofrHDL-DTPA-PE-Gd will be prepared in saline with different concentrationsfrom 0-3 mM in 1.0 ml centrifuge tubes. The samples will be placed in a30 mm diameter birdcage coil and placed in the 9.4 T MR system. Allexperiments will be performed at 37° C. T1 relaxation data will becollected with a spin-echo sequence with a variable TR (300, 800, 1500,2000, 3000 msec) and with a TE of 12.8 msec. T1 values of the sampleswill then be calculated from a 3 parameter exponential fit and plottedas 1/T1 vs. Gd concentration. The slope of this line will be molarrelaxivity, R1. This would be used to perform quality control and dosageadjustments.

As disclosed in US Pat Appl 20070243136 and incorporated herein byreference, interstitial penetration of the particles of the inventionreflects the traversal across endothelium (required for entry intoplaques). In order to assess this parameter, groups of young adult apoE-KO (n=15) and WT (n=15) mice are administered one of the following:¹²⁵I-labeled native human HDL (positive control), ¹²⁵I-labeled rHDL,¹²⁵I-labeled Gd-rHDL, or ¹²⁵I-human VLDL (too large to significantlycross the endothelium; negative control) (Sloop et al J Lipid Res 1987;28:225-37; Vessby et al. J Lipid Res 1987; 28:629-41). The lipoproteinsmay be labeled in either the apo A-I (HDL) or apoB (VLDL) moieties usinga standard iodination protocols (Fuki et al. J Clin Invest 1997;100:1611-22). To simulate the first series of imaging studies, eachmouse will receive at least 10¹⁷ lipoprotein particles. The t_(1/2) forHDL/apoA-I in the circulation of a mouse is roughly 10 h (Tape C. &Kisilevsky R. Biochim Biophys Acta 1990; 1043:295-300). Thus, during the10th hour after injection (i.e., from 9 to 10 h after injection), asuction blister will be induced on the back of each mouse, following astandard procedure used extensively in rodents (and humans) to inducethe extravasation of interstitial fluid (e.g., see Vessby et al. J LipidRes 1987; 28:629-41; Herfst M. J. & van Rees H. Arch Dermatol Res 1978;263:325-34). At 10 h (i.e., about 1 half-life of native HDL) afterinjection, interstitial fluid will be harvested from each suctionblister, and then the mice will be sacrificed and plasma samples takenfor gamma counting. Based on the predicted physical properties of thenative and rHDL particles, both are expected to exhibit ratios ofinterstitial-to-plasma concentrations of approximately 1:3. In contrast,the labeled VLDL should be almost entirely excluded from theinterstitial space (Sloop et al. J Lipid Res 1987; 28:225-37; Vessby etal. J Lipid Res 1987; 28:629-41). If the Gd-rHDL particles exhibitsubstantially less interstitial penetration than ¹²⁵I-HDL, the sizeand/or surface charge of the reconstituted particles may requireadjustment.

In certain embodiments it may be desirable to monitor thebiodistribution, autoradiography, and metallic contrast ion content ofthe rHDL compositions of the present invention. In exemplary methods agroup of apo E-KO (n=15) and WT (n=15) mice can be fed on the well-known“Western diet” for 16 weeks (to accelerate the formation of lesions inthe aorta of apo E-KO mice). The imaging compositions of the presentinvention, e.g., rHDL-DTPA-PE can be labeled with ¹¹¹In(rHDL-DTPA-PE-¹¹¹In) using standard methods (Phillips W. T. Adv DrugDeliv Rev 1999; 37:13-32) and injected via the tail vein. After 4 h, 12hrs, and 24 hrs post injection, randomly selected mice (5 in each group)can be sacrificed and the following tissues harvested: blood, heart,lungs, liver, kidneys, spleen, stomach, bone, muscle, skin, urine andaorta.

The organs are weighed and the radioactivity counted. The organbiodistribution values is expressed as % injected dose/g tissue (%ID/g). The aorta should be subjected to further study. As lesions willbe diffusely distributed, half of the aorta is reserved for sectioningof lesioned and non-lesioned areas and the other half openedlongitudinally and stained with Sudan IV to visualize lesions.Autoradiographic images are obtained using routine techniques ofexposure of the stained aorta to Kodak Biomax high-speed film for adesired period of time, e.g., 1 week. By comparing the location of theautoradiographic signals to the Sudan staining, it is possible todetermine the presence of rHDL and if there is enrichment in lesionedvs. nonlesioned areas. In the absence of a targeting molecule (or otherstrategies to promote retention of the rHDL in lesions) detectable, butrelatively low, signals are expected, including at lesion sites. Anadvantage of the compositions of the present invention as compared tothe use of iron particles or small micelles, which also can enterlesions, but are retained “non-specifically,” the molecules of thepresent invention are specifically targeted to a specific site. Thus,while iron particles and small micelles produce only non-specificsignaling, the molecules of the present invention provide an excellentalternative for signal enhancement based on the presence of specificcells (e.g., macrophages) and/or molecules of interest within theplaque.

The portions of aorta reserved for sectioning may be processed usingmethods known to those of skill in the art, (e.g., Rong et al.Circulation 2001; 104:2447-52; Choudhury R. P. & Fisher E. A.Arterioscler Thromb Vasc Biol 2009; 29:983-91; Choudhury et al.Arterioscler Thromb Vasc Biol 2004; 24:1904-9; Fayad et al. Circulation1998; 98:1541-7), but with the addition here of autoradiography andimmunostaining for human apoA-I and or apo A-II. Human-specificmonoclonal antibodies against apoA-I and apo A-II for this purpose areknown to those of skill in the art. These data allow confirmation of theentry of the rHDL into the aortic wall and to determine its distributionwithin the tissue. These assays (on Sudan-stained intact aorta as wellas on aortic sections) may be repeated for the rHDL engineered topromote its retention in plaques.

In a parallel mouse study as above (15 apoE-KO and 15 WT), the Gdcontent in the aorta after sacrifice is assessed by inductively coupledplasma (ICP) analyses (Gailbraith Labs, Inc.) (Glogard et al. Int JPharm 2002; 233:131-40; Fossheim et al. Magn Reson Imaging 1999;17:83-9; Tamat et al. Pigment Cell Res 1989; 2:281-5).

As disclosed in US Pat Appl 20070243136 and incorporated herein byreference, methods of performing in vivo MRM also may be used to testthe efficacy of the present imaging compositions. Contrast agentinjection of rHDL-DTPA-PE-Gd into animals (15 apoE-KO; and 15 WT) and invivo MRM may be performed. Briefly, to assess the plaque the followingMRM may be conducted: 1) in vivo multicontrast black-blood pre-contrastenhanced (CE) MRM; 2) in vivo postcontrast (rHDL-DTPA-PE-Gd complex) CEMRM black-blood T1W imaging; and 3) ex vivo post-CE MRM. Multipledifferent contrast agent concentrations should be tested based on therelaxivity and biodistribution results. As noted herein, initialinjections should preferably contain ˜10¹⁸ Gd ions, delivered asrHDL-Gd-DPTA-PE in an injection volume of ˜0.3 ml per mouse.

Following in vivo experiments, ex vivo MRM also may be performed. Theanimals are euthanized, and perfused fixed at physiological pressure.The heart is harvested and fixed in 4% paraformaldehyde for at least 24hr. Ex vivo MRM experiments are performed using a 10-mm birdcage coilusing methods previously described (Itskovich et al. Magn Reson Med2003; 49:381-5; Fatterpekar et al. AJNR Am J Neuroradiol 2002;23:1313-21). Briefly, each specimen will be washed and placed in an 8-mmpolyethylene tube filled with Fomblin (perfluoropolyether, Ausimont USAInc., Morristown, N.J.) and sealed to prevent air bubbles. The use ofFomblin limits tissue dehydration and MR artifacts on the surface of thespecimen. Multicontrast MR images are acquired with same parameters asin the in vivo sequence but now with a 25-50 um/pixel resolution. Thespecimens may further be histologically analyzed.

The apparati for use in imaging tissues are not considered limiting tothe invention and the compositions may be used in any MRI techniqueknown to those of skill is the art. Simply by way of example, those ofskill in the art referred to e.g., U.S. Pat. Nos. 6,590,391; 6,591,128;6,586,933; 6,580,936; 6,600,401; 6,611,143; and 6,541,973, whichdescribe MRI apparati in detail. These are merely exemplary descriptionsand those of skill in the art will be aware that other imagingtechniques may be used in other to perform the methods of the presentinvention using the compositions described herein.

The methods described in U.S. Pat. No. 6,498,946 may be particularlyuseful in magnetic resonance microscopy (MRM) and atherosclerotic plaqueimaging as discussed infra. In particular embodiments, the compositionsof the present invention may be used to assess the efficacy or dosing ofa particular existing drug. For example, in the case of atherosclerosis,the atherosclerotic lesion size or composition may be monitored prior toand after the administration of a given drug treatment to assess whetherthe treatment is effective at reducing the size or composition of alesion.

In particularly preferred embodiments, the compositions of the presentinvention can be used for targeting macrophages in imaging ofmacrophage-related diseases characterized by neoplastic tissue (US PatAppl 20010002251) including, but not limiting to, the cancers sarcoma,lymphoma, leukemia, carcinoma and melanoma, cardiovascular diseases(e.g., arteriosclerosis, atherosclerosis, intimal hyperplasia andrestenosis) and other activated macrophage-related disorders includingautoimmune diseases (e.g., rheumatoid arthritis, Sjogrens, scleroderma,systemic lupus erythematosus, non-specific vasculitis, Kawasaki'sdisease, psoriasis, Type I diabetes, pemphigus vulgaris), granulomatousdiseases (e.g., tuberculosis, sarcoidosis, lymphomatoid granulomatosis,Wegener's granulomatosus), inflammatory diseases (e.g., inflammatorylung diseases such as interstitial pneumonitis and asthma, inflammatorybowel disease such as Crohn's disease, and inflammatory arthritis), andin transplant rejection (e.g., in heart/lung transplants). Examples ofmacrophage-related diseases are also macrophage-related pulmonarydiseases such as emphysema (Marten K. & Hansell D. M. Eur Radiol 2005;15:727-41; US Pat Appl 20050281740).

H. Pharmaceutical Compositions and Kits Comprising rHDL

It is contemplated that that rHDL compositions of the invention will beused in MRI or other imaging method in any in which it is desired toobtain an image of an internal site. Thus, the compositions of theinvention will be administered, in vivo. Therefore, it will be desirableto prepare the compositions of the invention as a pharmaceuticalcomposition appropriate for the intended application. Generally thiswill entail preparing a pharmaceutical composition that is essentiallyfree of pyrogens, as well as any other impurities that could be harmfulto humans or animals. One also will generally desire to employappropriate salts and buffers to render the complex stable and allow forcomplex uptake by target cells.

As disclosed in US Pat Appl 20070243136 and incorporated herein byreference, aqueous compositions of the present invention comprise aneffective amount of the rHDL to deliver an appropriate amount ofmetallic or non-metallic contrast agent, dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. Such compositionscan also be referred to as inocula. The phrases “pharmaceutically orpharmacologically acceptable” refer to molecular entities andcompositions that do not produce an adverse, allergic or other untowardreaction when administered to an animal, or a human, as appropriate. AsHDL is normally found circulating in the system of an animal, it iscontemplated that the rHDL imaging compositions of the invention shouldnot produce such an adverse effect. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents and the like. The use of such media and agents forpharmaceutical active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with the rHDLof the invention (e.g., so long as the agent does not destroy thestructural integrity of the molecule or quench the signal of themetallic ion), its use in the compositions of the invention iscontemplated. Supplementary contrast enhancing ingredients also can beincorporated into the compositions.

The term “effective amount” as used herein refers to any amount of therHDL compositions of the invention that produce a reproducible andevaluable image of a given in vivo site. Thus, it should be understoodthat the effective amount of the rHDL may vary depending on the size ofthe animal, the site at which the composition is to be administered andthe route of such administration. The field of MRI technology isadvanced and technicians are experienced in determining whether a givencomposition is producing the desired intensity of signal. In specificembodiments discussed in the examples, it was determined that 10Gd-DPTA-PE molecules/rHDL particle may be an exemplary dose to imageatherosclerotic plaques in mice. It should, however, be understood thatmore or less than 10 Gd-DPTA-phospholipid molecules/rHDL particle alsoare contemplated. For example, the imaging compositions mayadvantageously comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 or more Gd-DPTA-phospholipid particles/rHDLmolecule. It should be understood that the number ofGd-DPTA-phospholipid/rHDL particle is limited only by how many suchmolecules can be incorporated into the rHDL particle without destroyingthe structural integrity of the HDL or making it larger than would beeffective as an imaging compositions of the invention (i.e., larger than18 nm.) With respect to the administration of rHDL, and using the apoAIcontent of the rHDL as a measuring parameter, it is contemplated thatthe amounts of rHDL administered to mice may comprise 8 mg, 10 mg, 12mg, 14 mg, 16 mg, 18 mg, 20 mg, 22 mg, 24 mg, 26 mg, 28 mg, or even 30mg of apoAI. Such compositions can be extrapolated to humans. Forexample, infusion of rHDL compositions of the present invention thatcomprise 45 mg apoA-I protein (or equivalent amount of apo A-II and/orpeptide fragments of apo A-I and A-II)/kg body weight of animal to betreated will be particularly useful. The compositions may thus comprise40 mg apoA-I protein/kg, 45 mg apoA-I protein/kg, 50 mg apoA-Iprotein/kg, 55 mg apoA-I protein/kg, 60 mg apoA-I protein/kg or more.Given the characteristics of HDLs discussed herein above, those of skillin the art should readily be able to determine the amounts of other HDLcomponents being administered in a given dose.

In certain embodiments, the amounts of phospholipid doses that can beused in the compositions described herein may be inferred fromexperience in administering Intralipid™ (Kabivitrum Inc., California andStockholm, an aqueous suspension of lipid droplets that is sterile andsuitable for intravenous feeding of patients. Other similar lipidsolutions that may provide guidance as to amounts and proportions oflipids that may safely be provided to patients include Nutralipid™(Pharmicia, Quebec), Liposyn™ (Abbot Labs, Montreal)). Thesecompositions are used by the biomedical optics community as a scatteringmedia in optical experiments. In other embodiments, the amount of rHDLthat is administered in the imaging embodiments of the present inventionis guided by the typical concentrations of HDL in human plasma, and thedose used in the imaging modalities described herein may use up todouble the concentration of HDL normally found in human plasma. Those ofskill in the art are aware of amounts of apo A-I and HDL that maytypically be administered. See e.g., U.S. Pat. Nos. 6,953,840;7,435,717; and 7, 491,693; Nissen et al. JAMA 2003; 290:2292-300, whichare incorporated herein by reference as showing parameters for theselection of patients that receive HDL and amounts of HDL that maytypically be administered. For example, the patients may receive Pane 85of 131 between 15 mg/kg or 45 mg/kg of rHDL. Those of skill in the artwill be able to vary the amount administered depending on the size andweight of the patient and the like.

The rHDL particles of the present invention are intended for use in anyMRI regimen that is conventional in the art, including MRI of humans.Administration of the imaging compositions according to the presentinvention will be via any common route used in imaging so long as thetarget tissue is available via that route. This includes administrationby orthotopic, intradermal subcutaneous, intramuscular, intraperitoneal,intrathecal, or intravenous injection. Alternatively, oral, nasal,buccal, rectal, vaginal or topical administration also are contemplated.For imaging atherosclerotic plaques intravenous injection iscontemplated. Such injections compositions would normally beadministered as pharmaceutically acceptable compositions that includephysiologically acceptable carriers, buffers or other excipients. Forimaging of tumors, direct intratumoral injection, injection of aresected tumor bed, regional (i.e., lymphatic) or general administrationis contemplated. It also may be desired to perform continuous perfusionover hours or days via a catheter to a disease site, e.g., a tumor ortumor site.

The imaging compositions of the present invention are advantageouslyadministered in the form of injectable compositions either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid prior to injection may also be prepared. The rHDLcompositions of the invention can be sterilized by heat, radiationand/or filtration, and used as such, or the compositions can be furtherdehydrated for storage, for instance by lyophilization.

The dehydrated material in form of a powder from which the MRI contrastagent may be produced by admixing the powder with a portion of carrierliquid and shaking. For practical application the compositions of theinvention in the medical field, it is contemplated that the driedcomponents and the carrier liquid can be marketed separately in a kitform whereby the contrast agent is reconstituted by mixing together thekit components prior to injection into the circulation of patients.

A typical composition for such purpose comprises a pharmaceuticallyacceptable carrier.

For instance; pharmaceutically acceptable carriers include aqueoussolutions, non-toxic excipients, including salts, preservatives, buffersand the like may be used. Examples of non-aqueous solvents are propyleneglycol, polyethylene glycol, vegetable oil and injectable organic esterssuch as ethyloleate. Aqueous carriers include water, alcoholic/aqueoussolutions, saline Pane 86 of 131 solutions, buffered solutions,parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.Intravenous vehicles include fluid and nutrient replenishers.Preservatives include antimicrobial agents, anti-oxidants, chelatingagents and inert gases. The pH and exact concentration of the variouscomponents the imaging composition may be adjusted according to wellknown parameters the amount and degree of signal intensity observed andrequired.

The individual components of the rHDL imaging compositions of thepresent invention may be provided in a kit, which kit may furtherinclude instructions for formulating and/or using the imaging agents ofthe invention. Such a kit will comprise a first composition comprising ametallic or non-metallic contrast agent, a second composition comprisinga phospholipid covalently linked to a chelating moiety, a thirdcomposition comprising HDL modified apolipoproteins A-I and A-II and/orfragments thereof, a fourth composition comprising a free phospholipid,and a fifth composition comprising a sterol. The kit may furthercomprise a sixth composition comprising HDL core lipids (e.g.,cholesteryl ester, and TG).

The kit also may comprise a device for delivering the composition to amammal.

EXAMPLES

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

The following non-limiting Examples are put forth so as to provide thoseof ordinary skill in the art with illustrative embodiments as to how thecompounds, compositions, articles, devices, and/or methods claimedherein are made and evaluated. The Examples are intended to be purelyexemplary of the invention and are not intended to limit the scope ofwhat the inventor regard as his invention. Efforts have been made toensure accuracy with respect to numbers (e.g., amounts, temperature,etc.) but some errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, temperature is in ° C.,or is at ambient temperature, and pressure is at or near atmospheric.

Example 1: Synthesis and Modification of Peptides

Paae 87 of 131

This example demonstrates one embodiment of a synthesized apo A-Ipeptide containing methionine residues at position 148 as referred tothe full-length apo A-I primary sequence. It also illustrates oneembodiment of a modified synthetic apo A-I peptide containing methioninesulfoxide at positions 148 as referred to the full-length apo A-Iprimary sequence. Although it is not necessary to understand themechanism of an invention, it is believed that being incorporated intothe rHDL compositions of the present invention, this modified peptidetargets the rHDL of the invention to sites of interest such, forexample, as macrophages in atherosclerotic plague.

It is well known to those of ordinary skill in the art that other apoA-I peptide fragments such as those containing methionine residues atpositions 86 or 112 can be easily synthesized using standard proceduresdescribed below or manufactured by any technique for peptide synthesisknown in the art. It is further understood by those of ordinary skill inthe art that methionine residues in these peptide fragments can beoxidized to methionine sulfoxides using the standard proceduresdescribed below (including those incorporated herein by reference) andthe modified peptides can be purified by any method known in the art,including HPLC. It should be also understood by those of ordinary skillin the art that apo A-I peptide fragments containing tyrosine residue atposition 192 can be easily synthesized or manufactured by any techniquefor peptide synthesis known in the art (including those incorporatedherein by reference). It is further understood by those of ordinaryskill in the art that tyrosine residue of these peptide fragments can beoxidized to 3-chloro-, 3-nitro- or 3,5-dibromotyrosine, using, forexample, the standard procedures known in the art (Shao et al. J BiolChem 2005; 280:5983-93; Weiss et al. Science 1986; 234:200-3). It iswell known to those of ordinary skill in the art that apo A-II peptidefragments containing methionine residue at position 148 as referred tothe full-length apo A-II primary sequence, can be easily synthesizedusing standard procedures described below or manufactured by anytechnique for peptide synthesis known in the art (including thoseincorporated herein by reference). It is further understood by those ofordinary skill in the art that methionine residues in these peptidefragments can be oxidized to methionine sulfoxides using the standardprocedures described below (including those incorporated herein byreference) and the modified peptides can be purified by any method knownin the art, including HPLC.

The first step is to synthesize the peptide corresponding to a portionof an apo A-I sequence. Although it is not necessary to understand themechanism of an invention, it is believed that being incorporated intothe rHDL compositions of the present invention, this peptide mimicsstructural and functional properties of full-length apo A-I.

The synthesis of peptides may involve the use of protecting groups.Peptides can be synthesized by linking an amino group to a carboxylgroup that has been activated by reaction with a coupling agent, such asdicyclohexylcarbodiimide (DCC). The attack of a free amino group on theactivated carboxyl leads to the formation of a peptide bond and therelease of dicyclohexylurea. It can be necessary to protect potentiallyreactive groups other than the amino and carboxyl groups intended toreact. For example, the .alpha.-amino group of the component containingthe activated carboxyl group can be blocked with a tertbutyloxycarbonylgroup. This protecting group can be subsequently removed by exposing thepeptide to dilute acid, which leaves peptide bonds intact.

In one embodiment, the amino acid sequence of a peptide comprisesNH₂-LQEKLSPLGEEMRDRARAHVDALRTHLAPY-OH (SEQ ID NO:1), hereafter referredto as “apo A-I Met148 peptide”.

An unprotected peptide can be synthesized or manufactured by anytechnique for peptide synthesis known in the art, including, e.g., thetechniques described in U.S. Pat. Nos. 6,004,925, 6,037,323 and6,046,166 with greater than 95% purity as assessed by HPLC.Alternatively, an unprotected peptide can be purchased from companiessuch as AnaSpec (Fremont, Calif., USA).

Peptide molecular mass can be checked by matrix-assisted laserdesorption ionization mass spectrometry.

To convert methionine in apo A-I Met148 peptide to methionine sulfoxide,the standard procedure known in the art to prepare apo A-I containingmethionine sulfoxides can be used (Anantharamaiah et al. J Lipid Res1988; 29:309-18; Sigalov A. B. & Stern L. J. FEBS Lett 1998;433:196-200; Sigalov A. B. & Stern L. J. Chem Phys Lipids 2001;113:133-46; von Eckardstein et al. J Lipid Res 1991; 32:1465-76).Briefly, a purified (about 15 mg) can be dissolved in 1 ml of 3 Mguanidine-HCl, pH 7.4, and then hydrogen peroxide was added to a finalconcentration of 300 mM. The mixture is incubated at 20° C. for 15 min,and an oxidized peptide is purified by preparative HPLC using aBioCAD/SPRINT System from PerSeptive Biosystems (Cambridge, Mass., USA),a Vydac C-18 column (22 mm×250 mm) and a two-solvent system: A,trifluoroacetic acid/water (1:1000, v/v); B, trifluoroaceticacid/acetonitrile/water (1:900:100, v/v). The column is heated to 50° C.in a water bath and peptides (modified and unmodified) are eluted at aflow rate of 15 ml/min with 28-49%, 49-53% and 53-73% gradient steps ofsolvent B over 12, 9 and 12 min, respectively. Then the content ofsolvent B is increased to 100% over 3 min, and finally decreased to 28%over 2 min. Peaks are identified by analytical HPLC. Analytical HPLC isperformed using a Waters Automated Gradient Controller, a Waters 745BData Processor and a Thermo Separation Products Spectra 100 UV-visibledetector, coupled to a Vydac C-18 column (4.6 mm×250 mm) heated to 50°C. and eluted with the same two-solvent system at a flow rate of 1.2ml/min and 28-64% gradient of B over 33 min. Then the content of B wasincreased to 100% over 2 min, and finally decreased to 28% over 2 min.The HPLC column eluates are monitored by absorbance at 214 nm. Massspectra of a purified modified peptide is measured using a Voyager EliteSTR mass spectrometer from PerSeptive Biosystems (Cambridge, Mass.,USA). As expected, conversion of one methionine residue to methioninesulfoxide results in increasing the molecular weight of the peptide by16 atomic mass units corresponding to an addition of one extra oxygenatom to the peptide molecule.

Example 2: Isolation and Purification of Apolipoproteins A-I and A-II

As discussed herein throughout, the present invention is related toimaging compositions that comprise rHDL as a backbone structure. Theapolipoproteins for the production of the rHDL composition may bederived from an animal as a source of the apolipoproteins for theproduction of the rHDLs. In a preferred method for the producing rHDLsthe apolipoproteins are from human HDL.

To isolate and purify human apolipoproteins A-I and A-II the standardprocedure known in the art can be used (Sigalov et al. J Chromatogr1991; 537:464-8). Briefly, HDL of density 1.063-1.210 g/ml were isolatedfrom fasting serum of normo-lipidaemic donors by sequentialultracentrifugation in a Beckman (Berkeley, Calif., U.S.A.) Model L8-7Qultracentrifuge using a 45.Ti rotor. The isolated HDL fraction wasextensively dialysed against 50 mM ammonium hydrogencarbonate buffer (pH8.2), lyophilized and delipidated by an original procedure using achloroform-methanol-diethyl ether solvent system as disclosed in(USSR/RUSSIA Pat 1752187), the disclosure of which is incorporatedherein by reference. The proteins were solubilized in 10 mM Tris-HClbuffer (pH 8.6) containing 8 M urea (Tris-urea buffer) and applied to aToyopearl HW-55F (Toyo Soda, Tokyo, Japan) column (90.0×3.5 cm I.D.).Elution was carried out with the same buffer at a flow-rate of 40 ml/hand 8-ml fractions were collected. A typical gel filtration profile isshown in FIG. 6A. Following analysis by polyacrylamide gelelectrophoresis (PAGE), fractions containing apo A-I and apo A-II werepooled (FIG. 6A). The apolipoprotein pool was applied to aDEAE-Toyopearl 650M (Toyo Soda) column (40.0×3.2 cm I.D.), equilibratedwith Tris-urea buffer. Elution was performed with a linear gradient ofsodium chloride from 0.02 to 0.15 M in Tris-urea buffer (1000 ml totalgradient volume) at a flow-rate of 60 ml/h. Fractions of 6 ml each werecollected. A typical ion-exchange profile is shown in FIG. 6B. Thosecontaining apo A-T and apo A-II were pooled separately (FIG. 6B) andextensively dialysed against 50 mM ammonium hydrogencarbonate buffer (pH8.2). After dialysis, the samples were desalted by gel permeationchromatography using a Toyopearl HW-40F (Toyo Soda) column (70.0×2.2 cmI.D.) with the same buffer at a flow-rate of 80 ml/h and finallylyophilized.

Example 3: Characterization of Apolipoproteins A-I and A-II

Apolipoproteins A-I and A-II isolated and purified as described inExample 2 were quantified according to Lowry el al. (Lowry et al. J BiolChem 1951; 193:265-75) and spectrophotometrically at 280 mm usingextinction coefficients of 1.22 and 1.82 AU/mg protein/ml for apo A-Iand apo A-II, respectively (Edelstein et al. J Biol Chem 1972;247:5842-9). Lipid phosphorus analysis was performed utilizing themethod of Bartlett (Bartlett G. R. J Biol Chem 1959; 234:466-8). Nophospholipid was detected in either the isolated apo A-I or apo A-II.Homogeneity was confirmed using the standard procedures well known inthe art such as SDS-PAGE on 15% polyacrylamide gels under both reducing(FIG. 7) and non-reducing conditions and by urea-PAGE. Identification ofapo A-I and apo A-II was confirmed by electrophoretic mobility andimmunoelectrophoresis with monospecific antibodies to human apo A-I andapo A-II (Clarke H. G. & Freeman T. Clin Sci 1968; 35:403-13). Aminoacid analyses of purified apo A-I and apo A-II were made in a Beckman6300 amino acid analyser after 72 h of acid hydrolysis and were shown tobe compatible with the compositions derived from the primary sequencesof human apo A-I and A-II well known in the art. The real yields ofpurified proteins were ˜50% and ˜40% for apo A-I and apo A-II,respectively, considering the volume of the initial human serum and theaverage concentrations of the proteins in the serum used (Sigalov et al.J Chromatogr 1991; 537:464-8).

To determine apo A-I concentration in humnan serum, thenon-immunochemical electrophoretic apo A-1 assay using pure andwell-characterized apo A-I as a primary standard can be used (Sigalov A.B. Eur J Clin Chem Clin Biochem 1993; 31:579-83). This is a precise,specific, and reproducible technique utilizing SDS-gradient(g)PAGE fordirect apo A-I measurement in human serum that does not depend on anyimmunoreaction and therefore avoids the problems associated withantibody-antigen interaction (the heterogeneity of antigenic sites inapo A-I, heterogeneity in their expression, and heterogeneity ofantibodies raised against apo A-I). The assay is inexpensive andrequires no radioisotopes, and the method possesses intrinsically highrecovery, due to minimal sample manipulation. No special samplepretreatment is required, and serum samples subjected to freezing orlyophilization can be used without any statistically significant changein results. Briefly, electrophoresis was performed in 18 cm×16 cm×1.5 mmgels with a linear gradient gel of 15-20% acrylamide, using homemadeapparatus. The stacking gel consisted of 3.75% acrylamide, 0.125 mol/1Tris-HCl, 1 g/l SDS, pH 6.8. A 1.5-mm-thick 17-well comb was used in allexperiments. Resultant wells were 5.0 mm wide and 2.0 cm high, allowingup to 60 ul of sample to be applied per line. Sample preparation,electrophoresis, gel staining and destaining were performed as described(France et al. J Lipid Res 1989; 30:1997-2004). A typical non-reducingSDS-gPAGE of 0.5, 1.0 and 2.0 ug of apo A-I (lanes 1-3), and fresh(lanes 4-7), frozen (lanes 8-13) and lyophilized (lanes 14-17) differentserum samples is shown in FIG. 8. After gel destaining the apo A-I bandswere excised and placed into 3.0-ml glass screw-top vials containing 1.0ml distilled water-dimethylformamide (1+1, by volume). Vials were heatedat 90° C. in a heating block for 1 h with periodic mixing. Theabsorbance was measured at 590 nm in a disposable semi-micro cuvette(Bio-Rad Laboratories, Richmond, Calif.) with a Spectronic-2000spectrophotometer (Bausch and Lomb, USA) blanked against distilledwater-dimethylformamide (1+1, by volume). Typical calibration curvesprepared with fresh, frozen and lyophilized samples of the pooled serumare shown in FIG. 9.

Example 4: Oxidative Modification of Apolipoprotein A-I and Reduction ofMethionine Sulfoxides by PMSR

This example demonstrates one embodiment of naturally occurring oxidizedapo A-I (apo A-I_(ox)) containing methionine sulfoxides at positions 112and 148 as referred to the apo A-I primary sequences. Although it is notnecessary to understand the mechanism of an invention, it is believedthat being incorporated into the rHDL compositions of the presentinvention, this modified protein targets the rHDL of the invention tosites of interest such, for example, as macrophages in atheroscleroticplague. The example also demonstrates that oxidative damage to apo A-Ican be reversed by PMSR in the presence of a source of reducingequivalents.

As described herein, apo A-I_(unox) refers to unoxidized apo A-Icontained in initial serum apo A-I and apo A-II_(unox) is unoxidized apoA-II contained in initial serum apo A-II. Further, apo A-I_(ox) isoxidized apo A-I (with sulfoxidized methionines at positions 112 and148) contained in serum apo A-I or obtained from unoxidized apo A-Iusing hydrogen peroxide. Apo A-I_(red) is reduced apo A-I obtained byreduction of oxidized apo A-I (with sulfoxidized methionines atpositions 112 and 148) using PMSR. In preferred embodiments, rHDL-1 arereconstituted HDL particles containing only apo A-I_(unox). In preferredembodiments, rHDL-2 are reconstituted HDL particles containing only apoA-I_(ox). In preferred embodiments, rHDL-3 are reconstituted HDLparticles containing apo A-I_(unox) and apo A-I_(ox) with a molar ratioof 1:1. In preferred embodiments, rHDL-4 are reconstituted HDL particlescontaining apo A-I_(unox), apo A-I_(ox) and apo A-II_(unox) with a molarratio of 3:3:1.

The initial apo A-I preparation isolated from pooled human serum andused in this example was a mixture of both apo A-I_(ox) (˜20%) and apoA-I_(unox) (˜80%) protein species, as shown by analytical reversed-phaseHPLC (FIG. 10A). This protein mixture migrated as a single band onSDS-PAGE (data not shown) and its chromatographic profile and retentiontimes of apo A-I_(ox) and apo A-I_(unox) were consistent with previouslyreported data (Anantharamaiah et al. J Lipid Res 1988; 29:309-18; vonEckardstein et al. J Lipid Res 1991; 32:1465-76). The percentage of apoA-I_(ox) in eight other apo A-I preparations obtained from pooled humansera (about 200 individual specimens in each pool) using the sameisolation technology had varied from 3% to 25%, providing furtherindirect evidence of considerable interindividual variability of theratio oxidized/unoxidized apo A-I (von Eckardstein et al. J Lipid Res1991; 32:1465-76). The two components (apo A-I_(ox) and apo A-I_(unox))in the initial serum apo A-I preparation were separated by preparativeHPLC. The isolated apo A-I_(unox) exhibited a single peak by analyticalHPLC (FIG. 10B) and a single band on SDS-PAGE (FIG. 11A, lane 1).

It is well known to those of ordinary skill in the art that the modifiedapo A-I molecules containing methionine sulfoxides at any one ofpositions 86, 112, 148, or any combination of said positions can beprepared and purified using the standard procedures described in thisinvention (including those incorporated herein by reference) and wellknown in the art. It should be also understood by those of ordinaryskill in the art that tyrosine residue at position 192 in apo A-I can beoxidized to 3-chloro-, 3-nitro- or 3,5-dibromotyrosine (and the modifiedprotein can be purified) using the standard procedures known in the art(Shao et al. J Biol Chem 2005; 280:5983-93; Weiss et al. Science 1986;234:200-3). It is well known to those of ordinary skill in the art thatmethionine residue at position 26 in apo A-II protein can be oxidized tomethionine sulfoxide using the standard procedures described in thisinvention (including those incorporated herein by reference) and themodified protein can be purified by any method known in the art,including HPLC. It should be also understood by those of ordinary skillin the art that methionine sulfoxides in any protein including but notlimiting to, apo A-I and apo A-II, can be reduced back to methioninenative form using PMSR and a source of reducing equivalents by thestandard procedures well known in the art and described in thisinvention (including those incorporated herein by reference) (see e.g.Biewenga et al. Arzneimittelforschung 1998; 48:144-8; Sigalov A. B. &Stern L. J. FEBS Lett 1998; 433:196-200; Sigalov A. B. & Stern L. J.Chem Phys Lipids 2001; 113:133-46; Sigalov A. B. & Stern L. J. AntioxidRedox Signal 2002; 4:553-7; Brot et al. Proc Natl Acad Sci USA 1981;78:2155-8).

To prepare apo A-I containing methionine sulfoxides at positions 112 and148, the standard procedure known in the art to can be used(Anantharamaiah et al. J Lipid Res 1988; 29:309-18; von Eckardstein etal. J Lipid Res 1991; 32:1465-76; Sigalov A. B. & Stern L. J. FEBS Lett1998; 433:196-200; Sigalov A. B. & Stern L. J. Chem Phys Lipids 2001;113:133-46; Sigalov A. B. & Stern L. J. Antioxid Redox Signal 2002;4:553-7). Briefly, the unoxidized protein (approx. 25 mg) was dissolvedin 1 ml of 3 M guanidine-HCl, pH 7.4, and then hydrogen peroxide wasadded to a final concentration of 300 mM. The mixture was incubated atroom temperature for 15 min, and an oxidized protein was purified usingpreparative HPLC. As has been shown (von Eckardstein et al. J Lipid Res1991; 32:1465-76), two methionine residues 112 and 148 (but not 86) inapo A-I are oxidized in parallel in a result of this procedure.

Treatment of apo A-I_(unox) with hydrogen peroxide resulted in theformation of apo A-I_(ox) which exhibited the same retention time as theapo A-I_(ox) component of the initial preparation (FIG. 10C). Theelectrophoretic pattern of purified apo A-I_(ox) on SDS-PAGE (FIG. 11A,lane 2) did not differ from that for the unoxidized form (FIG. 11A,lane 1) while on non-denaturing PAGE the oxidized form exhibited a bandwith about 1.8 times higher electrophoretic mobility in comparison witha relevant band of apo A-I_(unox).

Incubation of apo A-I_(ox) with purified PMSR in the presence of DTTresulted in the appearance of a new peak on analytical HPLC chromatogram(apo A-I_(red)) with the same retention time as for apo A-I_(unox) (FIG.10D). Similar results were obtained when a spinach S-30 cell-freeextract prepared as described (Brot et al. Proc Natl Acad Sci USA 1981;78:2155-8) was added as the source of the enzyme. Apo A-I_(re)d proteinformed as a result of enzymatic reduction was isolated as a homogeneouspreparation (FIG. 10E), having the same electrophoretic mobility as apoA-I_(unox) on SDS-PAGE (FIG. 11A, lane 3) and on nondenaturing PAGE.

Matrix-assisted laser desorption mass spectrometry revealed that apoA-I_(ox) was 32 mass units greater than the initial apo A-I_(unox) andapo A-I_(red). Peptide mapping data demonstrated that methionineresidues at positions 112 and 148 are sulfoxidized under theseconditions. Therefore, under the conditions used in this example, bothof the labile methionine residues Met-112 and Met-148 are oxidized byH₂O₂, consistent with previously reported data (von Eckardstein et al. JLipid Res 1991; 32:1465-76), and both are reduced by PMSR.

Importantly, naturally occurring apo A-I_(ox) purified from human serumand apo A-I_(ox) artificially obtained from unoxidized protein usingH₂O₂ represent the same apo A-I molecule containing methioninesulfoxides at positions 112 and 148.

To further confirm the presence of methionines and methionine sulfoxidesin apo A-I preparations, cleavage by CNBr can be used. CNBr cleavesproteins by conversion of methionine to homoserine lactone withconcomitant peptide bond cleavage, but methionine sulfoxides areresistant to reaction. Unoxidized and reduced apo A-I had the sameelectrophoretic pattern and were almost completely cleaved by CNBr whileapo A-I_(ox) was much more resistant (FIG. 11B). Oxidation of the twomethionine residues of apo A-I_(ox) results in a dramatic increase inthe protease susceptibility (FIG. 11C, lane 2) in comparison with theunoxidized and reduced forms (FIG. 11C, lanes 1 and 3).

Far-UV circular dichroism spectra and temperature-induced unfolding ofunoxidized, oxidized, and reduced apo A-I can be used to characterizestructural features of these proteins (FIGS. 12A-D). Briefly, circulardichroic spectra were measured for unoxidized (FIG. 12A), oxidized (FIG.12B) and reduced (FIG. 12C) apo A-I (7.2 uM) with (dotted line) andwithout (solid line) 2.3 mM 1,2-diheptanoyl-sn-glycero-3-phosphocholine(DHPC) or in the presence of 4 M urea (dashed line) in 10 mM ammoniumbicarbonate, 0.005% sodium azide, pH 7.8; in a 1 mm path-length cell at25° C., with 1 nm bandwidth and 1.0 s averaging per point.Temperature-induced unfolding data (FIG. 12D) were collected in atemperature range of 25−95° C. at 222 nm on a solution of 7.2 uM apo A-Iin 10 mM ammonium bicarbonate, 0.005% sodium azide, pH 7.8; in a 1 mmpath-length cell; with 1 nm bandwidth, 1° C. temperature increment, and5.0 s averaging per point. The spectrum of apo A-I_(ox) (FIG. 12B) wasconsiderably less intense than that of the unoxidized form (FIG. 12A),indicating a reduction of apo A-I helical content upon oxidation.Thermodynamically, apo A-I_(unox) was characterized by a weaklycooperative unfolding with midpoint temperature 65±2° C. indicative of aglobular, folded structure, while apo A-I_(ox) did not exhibit anycooperative unfolding transition, suggestive of a largely unfoldedstructure (FIG. 12D).

Thus, reduction of A-I_(ox) with PMSR in the presence of DTT completelyrestored protein secondary structural features and the characteristicthermal denaturation of the native unoxidized protein (FIG. 12C-D).

Alternatively, clinically relevant DHLA can be used instead of DTT as acofactor for PMSR-mediated reduction of methionine sulfoxides atpositions 112 and 148 in apo A-I_(ox) back to native methionine form(see e.g., Sigalov A. B. & Stern L. J. Antioxid Redox Signal 2002;4:553-7), restoring protein secondary structural features and thethermodynamic stability of the native unoxidized protein (Table 1).Thermodynamic stability of the proteins can be measured by the standardprocedures known in the art. Briefly, the effect of guanidinehydrochloride (GdnHCl) concentration on the structure of lipid-free apoA-I was monitored by fluorescence emission of Trp using a FluoroMax-2spectrofluorimeter (SPEX Industries, Inc., Edison, N.Y., U.S.A.).Denaturation curves were analyzed as previously described in detail(Sigalov A. B. & Stern L. J. Chem Phys Lipids 2001; 113:133-46).Stability of lipid-free apo A-I was also determined by plotting the 353nm/333 nm fluorescence emission ratio versus the molar GdnHClconcentration, and expressed as the concentration of the denaturant thatreduced this ratio by 50% (D_(1/2)).

TABLE 1 Secondary structure and thermodynamic stability of lipid-freeapo A-I ΔG_(D)°^(c) Δn^(d) D_(1/2) ^(b) (kcal/mol (mol alpha-Helix^(a)(M of GdnHCl/mol T_(m) ^(e) Protein (%) GdnHCl) apo) of apol) (° C.) apoA-I_(unox) 62 ± 4 1.0 ± 0.1 4.7 ± 0.3 29 ± 4 64 ± 3 apo A-I_(ox)  42 ±4^(f )  0.4 ± 0.1^(f )  1.4 ± 0.1^(f )  15 ± 3^(f ) — apo A-I_(red) ^(g)64 ± 4 0.9 ± 0.1 4.4 ± 0.3 27 ± 4 62 ± 3 Results are given as means ± SD(n = 3). ^(a)Determined from molar ellipticities at 222 nm.^(b)Midpoints of GdnHCl denaturation. ^(c)Free energy of denaturation atzero GdnHCl concentration. ^(d)The number of the GdnHCl moles boundduring denaturation. ^(e)Midpoints of thermal denaturation. ^(f)P <0.05, comparison versus apo A-I_(unox). ^(g)Reduced with DHLA as acofactor of PMSR.

Example 5: Methods of Reconstitution and Characterization of rHDL

The purified unmodified and modified apo A-I and A-II as well as peptidefragments thereof described in Examples 1-4 can then be reconstituted inany combination of said agents with two different sets of lipids. Asdisclosed in US Pat Appl 20070243136 and incorporated herein byreference, as the major circulating form of HDL is spherical, andspherical rHDL has been successful in delivering drugs to tissues, it ispreferable to reconstitute HDL as spherical particles. Thus, the firstof lipids in which the apolipoproteins may be reconstituted in arelipids that will produce spherical HDL particles. This set includessurface lipids, chiefly phospholipids, plus core lipids, chiefly TG andcholesteryl ester. This composition preferably should mimic natural HDLand generate small spherical particles (about 10 nm diameter).Particular reference is made to Rensen et al. (Rensen et al. Adv DrugDeliv Rev 2001; 47:251-76), for characteristics of circulating HDL thatcould be mimicked.

Alternatively, it may be desirable to produce a discoidal rHDL particle.This is achieved with the second set of lipids for reconstitution, whichis limited exclusively to surface lipids (i.e., preparing rHDLcomposition that lacks the core lipids). At high protein:lipid ratios,this composition generates small discoidal particles, which areessentially segments of lipid bilayers with the edges stabilized by theHDL apolipoproteins. These disks are about 10 nm in diameter and 5.5 nmthick and are thought to resemble nascent HDL as it is secreted from theliver. As discussed further below, discoidal reconstituted particles arean attractive alternative to spherical rHDLs in those circumstances inwhich the loading to the HDL particles with the imaging or targetingagent makes the spherical HDL particles too large (Shamir et al. J ClinInvest 1996; 97:1696-704). Discoidal particles can be made by a standardsonication method (Lund-Katz S. & Phillips M. C. Biochemistry 1986;25:1562-8) that has also been used for the incorporation of drugs (deVrueh et al. Antimicrob Agents Chemother 2000; 44:477-83).

Based on the weight % composition of the components of native HDL(Rensen et al. Adv Drug Deliv Rev 2001; 47:251-76) and the molecularweights of POPC (palmitoyl oleoyl PC), triolein (the triglyceridecomponent), cholesterol, cholesteryl oleate (cholesteryl ester) andapoA-I, the molar composition of the rHDL, should be(PC:CE:C:TG:apoA-I):100:62:25:11:2 to simulate the native particles(assuming apoA-I represents approximately 50% of HDL protein).Alternatively, equivalent amounts of apo A-II or peptide fragments apoA-I and apo A-II can be used in these preparations. For the compositionsof the present invention, it is critical that the rHDL prepared containat least one modified molecule of apo A-I and/or apo A-II and/orfragments thereof described in Examples 1-4. For ease of manufacture andstorage, these components, particularly POPC, were selected to berelatively resistant to lipid peroxidation, yet they remain fluid withinthe reconstituted particle at body temperature.

To produce reconstituted particles, the lipids (separately maintained instock solutions of chloroform or hexane) are combined in the appropriatemolar amounts in a glass tube and dried under nitrogen and then highvacuum to remove all traces of organic solvents. After suspension inTris pH 8.0 buffer, the lipids are dispersed by sonication, followed bylow speed centrifugation to remove any shards from the sonicator probetip. ApoA-I (in Tris pH 8.0 buffer) is then added (to 2 mol %) andincubated at 37° C. for 30 min, after which the mixture is againsonicated and re-centrifuged at low speed. The resulting dispersion isfiltered (0.22 um), and the rHDL isolated by size exclusionchromatography through a Superose 6 column. Typically, this procedureresults in spherical HDL particles of 7-9 nm diameter with approximately80-90 phospholipid molecules and 2 apoA-I molecules per particle(Braschi et al. J Lipid Res 1999; 40:522-32; Ramsamy et al. J Biol Chem2000; 275:33480-6). Extrusion, cholate dialysis, and/or shear methodse.g., using microfluidizers also can be used for reconstitution.

As PE is readily incorporated into liposomes (at least 50% by weight ofthe phospholipid content) (Grant et al. Magn Reson Med 1989; 11:236-43)and HDL (at least 10 mole %) (Lund-Katz et al., Biochemistry. 1986;25:1562-8), and owing to its close structural similarity to PC,Gd-DPTA-PE should readily be incorporated into the rHDL particles usingco-sonication (Lund-Katz S. & Phillips M. C. Biochemistry 1986;25:1562-8).

As disclosed in US Pat Appl 20070243136 and incorporated herein byreference, in preparing the rHDL compositions, it may be necessary toconsider is how much chelate should be incorporated into each HDLparticle. Administration of 10¹⁸ Gd ions per mouse is sufficient toobtain high-quality images of lesional aorta (Nunn et al. Q J Nucl Med1997; 41:155-62; Aime et al. J Magn Reson Imaging 2002; 16:394-406;Lauffer R. B. Magn Reson Q 1990; 6:65-84; Ahrens et al. Proc Natl AcadSci USA 1998; 95:8443-8). Using rHDL compositions of the presentinvention, this dose of Gd is easily accomplished by incorporating 10Gd-DPTA-PE molecules per rHDL particle and administering approx. 10¹⁷particles per mouse. To maintain particle structure, the Gd-DPTA-PEwould replace POPC, molecule for molecule. Based on the stoichiometrygiven in the preceding paragraph, 10¹⁷ rHDL particles containapproximately 8 mg of apoA-I and 13 mg of phospholipid. Because rHDL andphospholipid liposomes have been used to deliver far higher doses tomice (up to 13 mg of apoA-I) (Shah et al. Circulation 2001; 103:3047-50)and 30 mg PC per mouse (Williams et al. Arterioscler Thromb Vasc Biol2000; 20:1033-9), the dose of rHDL proposed for use here (i.e., 10Gd-DPTA-PE molecules/rHDL particle) should be readily achievable withouttoxicity.

Producing Discoidal rHDL

This example demonstrates one embodiment of the homogeneous discoidalrHDL prepared with or without naturally occurring oxidized apo A-Icontaining methionine sulfoxides at positions 112 and 148 as referred tothe apo A-I primary sequences. Although it is not necessary tounderstand the mechanism of an invention, it is believed that beingincorporated into the rHDL compositions of the present invention, thismodified protein targets the rHDL of the invention to sites of interestsuch, for example, as macrophages in atherosclerotic plague. The examplealso demonstrates that oxidative damage to apo A-I in the context ofrHDL can be reversed by PMSR in the presence of a source of reducingequivalents.

It should be understood by those of ordinary skill in the art that anyof the purified unmodified and modified apo A-I and A-II as well aspeptide fragments thereof described in Examples 1-4 can be used toproduce the compositions of the present invention. However, it iscritical for the rHDL of the invention that the the rHDL prepared shouldcontain at least one modified molecule of apo A-I and/or apo A-II and/orfragments thereof described in Examples 1-4.

In preferred embodiments, the homogeneous discoidal rHDL compositions ofthe present invention can be prepared using the standard sodium cholatedialysis method well known in the art (Sigalov A. B. & Stern, L. J. ChemPhys Lipids 2001; 113:133-46; Sigalov A. B. & Stern L. J. Antioxid RedoxSignal 2002; 4:553-7; Sorci-Thomas et al. J Biol Chem 1998;273:11776-82; Durbin D. M. & Jonas A. J Biol Chem 1997; 272:31333-9;Davidson et al. J Biol Chem 1995; 270:5882-90; Toledo et al. ArchBiochem Biophys 2000; 380:63-70). This method allows to prepare rHDLwith 2 or 3 apo A-I per particle and 9-11 nm diameter.

In preferred embodiments, rHDL-1 are reconstituted HDL particlescontaining only apo A-I_(unox). In preferred embodiments, rHDL-2 arereconstituted HDL particles containing only apo A-I_(ox). In preferredembodiments, rHDL-3 are reconstituted HDL particles containing apoA-I_(unox) and apo A-I_(ox) with a molar ratio of 1:1. In preferredembodiments, rHDL-4 are reconstituted HDL particles containing apoA-I_(unox), apo A-I_(ox) and apo A-II_(unox) with a molar ratio of3:3:1.

All preparations were done in Tris-buffered saline containing 0.01 MTris-HCl, 0.14 M NaCl, 0.25 mM EDTA-Na₂, 0.15 mM sodium azide, (TBS), pH7.4. Briefly, 65 ul of POPC in chloroform (about 2.0 mg) and 5.0 ul ofcholesterol in chloroform-ethanol, 1:1 (about 50 ug) were mixed in a 1.5ml polypropylene tube, dried in a stream of argon, and placed undervacuum for 30 min. Then, 100 ul of sodium cholate in TBS, pH 7.4, (about2.1 mg) was added and vortexed. After incubation for 30 min at 25° C.,280 ul of a solution containing about 0.9 mg apo A-I_(unox), A-I_(ox),or a mixture of apo A-I_(unox) and A-I_(ox) (molar ratio of 1:1), orinstead 1.1 mg of a mixture of apo A-I_(unox)-A-I_(ox)-A-II_(unox)(molar ratio of 1.5:1.5:1) were added. After additional incubation for90 min at 25° C., cholate was removed by dialysis against 1 l of TBS, pH7.4, for 1.5 h at room temperature and then against 3 l of the samebuffer for 16 h at 4° C. The obtained rHDL were then isolated on acalibrated Superdex 200 HR (10×300 mm2) gel filtration column(Pharmacia) eluted at 0.4 ml/min with TBS, pH 7.4. Molecular weight ofthe rHDL particles was calculated from their retention times relative togel filtration proteins standards supplied by Bio-Rad: thyroglobulin(670 000 Da), bovine gamma globulin (158 000 Da), chicken ovalbumin (44000 Da), equine myoglobin (17 000 Da), and vitamin B-12 (1350 Da). Theisolated rHDL samples were filtered through a 0.22 um using Spin-Xcentrifuge tubes (Corning Costar Corporation, Cambridge, Mass.) andstored at 4° C.

Characterization of Composition, Size and Shape of ReconstitutedDiscoidal rHDL

Reconstituted discoidal rHDL were characterized as described (Sigalov A.B. & Stern, L. J. Chem Phys Lipids 2001; 113:133-46). Proteinconcentrations in the rHDL particles were measured using the Lowrymethod as modified by Markwell et al. (Markwell et al. Anal Biochem1978; 87:206-10). Final protein compositions were determined in theprepared rHDL particles by analytical HPLC essentially as describedabove in Example 4 for lipid-free apolipoproteins, except that a solidGdnHCl was added to the analyzed rHDL samples to a final concentrationof 6 M. Total cholesterol was determined enzymatically using aBoehringer-Mannheim kit and the manufacturer's suggested procedure.Phospholipids were determined by phosphorus assay (Van Veldhoven P. P.and Mannaerts G. P. Anal Biochem 1987; 161:45-8). The number of apo A-Imolecules per particle was determined by cross-linking performed byaddition of one part dimethylsuberimidate (DMS) solution, 10 mg/ml in1.0 M triethanolamine, pH 9.7, to ten parts rHDL solution, incubationfor 2 h at 25° C. (Swaney J. B. Methods Enzymol 1986; 128:613-26) and bydetermination of extent of oligomer formation using SDS-PAGE (12.5%acrylamide). The gels were stained for protein with Coomassie Blue R250and scanned with an Hewlett Packard ScanJet 3P. The obtained images wereanalyzed using a NIH Image 1.61 program (National Institutes of Health,Bethesda, Md.) and a Scion Image 3b program (Scion Corporation,Frederick, Md.).

The sizes and size distributions of the rHDL particles were estimated byboth electron microscopy (EM) and nondenaturing gradient gelelectrophoresis (GGE). The rHDL complexes (at a concentration of about0.3 mg of protein/ml) were extensively dialyzed against 5 mM ammoniumbicarbonate, mixed with the same volume of 2% phosphotungstate, pH 7.4,and were examined using a Phillips EM410 electron microscope oncarbon-coated Formvar grids. Microphotographs were photographed at aninstrument magnification of 63000 and 110000, and mean particledimensions of 50 particles were determined from each negative.Nondenaturing GGE was performed on precast 4-20% gradient gels (Bio-Rad,Hercules, Calif.). Gel scanning and image analysis were performed asdescribed above. Stokes' diameter and molecular weight of the rHDLparticles were calculated from their mobility relative to proteinsstandards supplied by Pharmacia: thyroglobulin (17.0 nm, 669000 Da),ferritin (12.2 nm, 440000 Da), catalase (10.4 nm, 232000 Da), lactatedehydrogenase (8.2 nm, 140000 Da), and bovine serum albumin (7.1 nm,67000 Da). The protein and lipid compositions together with the apo A-Ioligomer size determined by cross-linking and the particle molecularweight were used to estimate the particle molar composition.

In preferred embodiments, compositions and properties of reconstitutedHDL particles are those as described in Table 2. These data demonstratethat as it is critical for the compositions of the present invention interms of resembling with nascent HDL, that oxidation of two of threemethionine residues (Met-112 and 148) in apo A-I molecule does not leadto any significant differences between prepared rHDL complexes in theirlipid and protein compositions as well as more importantly, in theirsize or shape.

TABLE 2 Properties and compositions of rHDL particles^(a) Particlecomposition POPC-Chol- Particle Particle alpha- Protein/ Protein Proteincomposition diameter diameter Helix Complex (mol:mol:mol)^(a) (molarratio)^(b) (EM; nm)^(c) (GGE; nm)^(d) (%)^(e) apo A-I_(unox) 62 (4) apoA-I_(ox) 42 (4) rHDL-1 180 (16):5 (1):3 Only A-I_(unox) 10.2 (1.0) 9.4(0.7) 78 (5) rHDL-2 180 (14):3 (1):3 Only A-I_(ox) 10.3 (1.0) 9.6 (0.5)81 (5) rHDL-3 190 (19):5 (1):3 A-I_(unox)-A-I_(ox) (1:1)^(f)  9.9 (1.0)9.6 (0.6) 82 (5) rHDL-4 150 (22):4 (1):3.5 A-I_(unox)-A-I_(ox):A-II(3:3:1)^(f) 10.3 (1.0) 9.6 (0.8) 78(5) ^(a)Mean and standard deviation(SD, in parentheses) of three different preparations are given. Thenumber of apolipoprotein molecules per particle was obtained bycross-linking with DMS (Swaney J. B. Methods Enzymol 1986; 128:613-26)and protein analysis by SDS-PAGE. ^(b)Determined by the reversed-phaseHPLC. ^(c)Mean and SD of 50 particles determined from negative stainingelectron microscopy (EM). ^(d)Mean and SD of three differentpreparations determined from nondenaturing gradient gel electrophoresis(GGE) using reference globular proteins. ^(e)Mean and SD of threedifferent preparations determined from molar ellipticities at 222 nm.^(f)Average compositions that may reflect subpopulations with differentmolar composition.Characterization of Protein Secondary Structure in ReconstitutedDiscoidal rHDL

CD spectra were collected on solutions of 3.6 uM (0.1 mg/ml) lipid-freeapo A-I proteins and of 1.8 uM (0.05 mg of protein/ml) rHDL particles inTBS, pH 7.4 with a 1 mm path-length quartz cuvette using an AVIV 62A DSspectropolarimeter (AVIV, Lakewood, N.J.). Data were collected at 25° C.every nanometer from 190 to 260 nm with 1.0 s averaging per point and a1 nm bandwidth. Spectra of at least six scans were signal averaged andbaseline corrected by subtracting an averaged buffer spectrum. Thespectra were normalized to molar residue ellipticity using a meanresidue weight of 115.2 and 113.6 Da for human apo A-I and A-II,respectively. An apparent fractional percent alpha-helix content fromthe molar ellipticities at 222 nm by the method of Chen et al.([ϕ]222=−30300, f_(H)=2340, where f_(H) is the fraction of alpha-helicalstructure; Chen et al. Biochemistry 1972; 11:4120-31).

No significant differences were observed between the CD spectra of thefour rHDL complexes (FIG. 13). Thus, as it is important for thecompositions of the present invention in terms of resembling withnascent HDL, the secondary structure of oxidized apo A-I molecules inrHDL complexes remains similar to that of the unoxidized protein.

Characterization of Thermodynamic Stability of Reconstituted DiscoidalrHDL

Temperature-induced unfolding data were collected on solutions of 3.6 uM(0.1 mg/ml) lipid-free apo A-I and 1.8 uM (0.05 mg of protein/ml) rHDLparticles in TBS, pH 7.4, with a 1 mm path-length quartz cuvette at 222nm every 2-5° C. from 25 to 95° C. with 20.0 s averaging per point and a1 nm bandwidth. T_(m) values for the broad transitions were estimated bycurve fitting using a seven-parameter equation (Zarutskie et al.Biochemistry 1999; 38:5878-87).

The effect of GdnHCl concentration on the structure of lipid-free andlipid-bound apo A-I was monitored by fluorescence emission of Trp asdescribed previously (Tricerri et al. Biochim Biophys Acta 1998;1391:67-78). These measurements were made in a FluoroMax-2spectrofluorimeter (SPEX Industries, Inc., Edison, N.Y.) at 25° C. in4×4 mm2 cuvette. Emission spectra were taken by exciting at 285 nm witha resolution of 2 nm and by measuring the emission with a resolution of4 nm. The molar GdnHCl concentrations (C) were determined from thesolution refractive index (n) using the relationship: C=60.87n-81.16, asdescribed (Kielley W. W. & Harrington W. F. Biochim Biophys Acta 1960;41:401-21). Aliquots of each lipid-free apo A-I protein (0.1 mg/ml) orrHDL sample (0.05 mg of protein/ml) were incubated with from 0 to 3.0 MGdnHCl (lipid-free proteins) or from 0 to 6.0 M GdnHCl (rHDL complexes)in TBS, pH 7.4, for 72 h at 4° C. Then, the Trp fluorescence spectrawere taken as described (Sigalov A. B. & Stern L. J. Chem Phys Lipids2001; 113:133-46) and the ratio of the fluorescence intensity at 353 nmto that at 333 nm was used to quantify the spectral shifts. Denaturationcurves were analyzed essentially as described (Sparks et al. J Biol Chem1992; 267:25839-47). Briefly, the following relationship between thefree energy of denaturation (ΔG_(D)) and the GdnHCl activity (a) wasused to estimate apo A-I conformational stability: ΔG_(D) ⁰=ΔG_(D)+Δn×RTln(1+Ka) (1) where ΔG_(D) ⁰ is the standard free energy of denaturation(at zero denaturant concentration), R is gas constant (1.98 cal/degreemol), T is temperature (298 K), K is average association constant ofGdnHCl and protein (0.6 M⁻¹), and Δn is the difference in the moles ofthe denaturant bound by the protein in the native and denatured states.The mean GdnHCl ionic activities (a) were calculated by the equation(Pace C. N. & Vanderburg K. E. Biochemistry 1979; 18:288-92):a=0.6761M+0.1468M+0.02475M³+0.001318M⁴ (2) where M is the molar GdnHClconcentration. The equilibrium constants, K_(D), were calculated fromthe 353 nm/333 nm fluorescence intensity ratios using the formula:K_(D)=([F]_(N)−[F])/([F]−[F]_(D)) (3) where [F] is the observed 353nm/333 nm fluorescence intensity ratio at a given GdnHCl activity and[F]_(N) and [F]_(D) are the 353 nm/333 nm fluorescence intensity ratiosfor the native and fully denatured forms of apo A-I measured in theabsence or presence of 3 M GdnHCl (lipid-free proteins) or 6.0 M GdnHCl(rHDL complexes). Linear regression analysis was used to solve Eq. (1)and to determine ΔG_(D) ⁰ and Δn. Stability of lipid-free andlipid-bound apo A-I was also determined by plotting the 353 nm/333 nmfluorescence intensity ratio vs the molar GdnHCl concentration andexpressed as the concentration of the denaturant that reduced this ratioby 50% (D_(1/2)).

No substantial differences in stability of apo A-I were observed amongthe four apo A-I-carrying rHDL complexes, including rHDL-4 which alsocontains apo A-II (Table 3, FIGS. 14-16). Thus, as it is important forthe compositions of the present invention in terms of resembling withnascent HDL, the structural stability of oxidized apo A-I molecules inrHDL complexes remains similar to that of the unoxidized protein.

TABLE 3 Thermodynamic parameters of lipid-free and lipid-associated apoA-I D_(1/2) ΔG_(D)° Δn (M (kcal/mol of (mol GdnHCl/mol T_(m) ComplexGdnHCl)^(a) apolipoprotein)^(b) of apolipoprotein)^(c) (° C.)^(d) apoA-I_(unox) 1.0 (0.1) 4.7 (0.3) 29(4) 64(3) apo A-I_(ox) 0.4 (0.1)^(f)1.4 (0.1 )^(f) 15 (3)^(f) — rHDL-1 4.1 (0.3)^(f) 3.7 (0.3)^(f) 7(1)^(f)82 (3)^(f, g) rHDL-2 3.3 (0.3)^(f, g) 3.1 (0.3)^(f, g) 7(1)^(f) 71(3)^(f, g) rHDL-3 3.8 (0.3)^(f) 3.0 (0.3)^(f, g) 6(1)^(f) 74 (3)^(f, g)rHDL-4e 3.7 (0.3)^(f) 3.2 (0.3)^(f, g) 7(1)^(f) 74 (3)^(f, g)^(a)Midpoints of GdnHCl denaturation. Mean and S.D. (in parentheses) ofthree different measurements are given. ^(b)Free energy of denaturationat zero GdnHCl concentration ( ± SD). ^(c)The number of the GdnHCl molesbound during denaturation ( ± SD). ^(d)Midpoints of thermal denaturation( ± SD). ^(e)Mean number of apolipoprotein moles calculated from thedata of reversed-phase HPLC. ^(f)P < 0.05, comparison vs apo A-I_(unox).^(g)P < 0.05, comparison vs rHDL-1.Limited Proteolytic Digestion of Reconstituted Discoidal rHDL

Samples of lipid-free apo A-I or rHDL complexes (0.4 mg of protein/ml)in PBS (pH 7.2) were treated with chymotrypsin at 37° C. for 75 minusing a protein-protease ratio (w/w) of 1000:1 (for lipid free apo A-I),or for 240 min using a ratio of 6:1 (for rHDL). Trypsin digestions wereperformed similarly, except in TBS (pH 7.4) for 120 min using aprotein-protease ratio of 200:1 (w/w). Digested samples were analyzed bySDS-PAGE (15% acrylamide) and HPLC.

Thus, the oxidative damage to the apo A-I molecule leads to theappearance of an accessible tryptic site in the central region of thelipid-bound apo A-I molecule (FIG. 17). These oxidation-induced changesin the accessibility of the apo A-I central region to proteolysis mayenhance the absorption of rHDL by macrophages in vivo which is criticalfor the compositions of the present invention.

Lipid Binding of Unoxidized, Oxidized and Enzymatically ReducedApolipoprotein

Unoxidized apo A-I, apo A-I_(unox), and oxidized apo A-I, apo A-I_(ox)(with sulfoxidized methionines at positions 112 and 148) were obtained,purified and characterized as described above (and in Sigalov A. B. &Stern L. J. FEBS Lett 1998; 433:196-200; Sigalov A. B. & Stern L. J.Chem Phys Lipids 2001; 113:133-46; Sigalov A. B. & Stern L. J. AntioxidRedox Signal 2002; 4:553-7). The rHDL complexes were prepared andcharacterized as described above (and in Sigalov A. B. & Stern L. J.Chem Phys Lipids 2001; 113:133-46; Sigalov A. B. & Stern L. J. AntioxidRedox Signal 2002; 4:553-7).

Enzymatic reduction of methionine sulfoxides in lipid-free apo A-I_(ox)(65-130 ug) and apo A-I_(ox) in rHDL particles (15 ug) was carried outessentially as described (Sigalov A. B. & Stern L. J. FEBS Lett 1998;433:196-200) at 37° C. in 33 mM Tris-HCl, 13 mM MgCl₂, pH 7.5,containing 4-8 ug of PMSR in a total volume of 30-90 ul, except thatclinically relevant DHLA (13 mM) was used as a cofactor of PMSR insteadof DTT. For HPLC analysis, solid GdnHCl was added to the reactionmixtures containing rHDL complexes to a final concentration of 6 M.

1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) in chloroformsolution was dried under argon and then solubilized in TBS(Tris-buffered saline containing 0.01 M Tris-HCl, 0.14 M NaCl, 0.25 mMEDTA-Na₂, 0.15 M NaN₃), pH 8.0 (0.5 mg/ml final) above its transitiontemperature (>24° C.). DMPC/protein molar ratios of 50:1 were used, andthe reaction was followed at 24° C. in a thermostated cell compartmentof a Hitachi U-3110 spectrophotometer by monitoring the decrease inabsorbance at 325 nm. The data were analyzed according topseudo-first-order kinetics, and t_(1/2) was determined as the timerequired for a 50% decrease in turbidity.

The ability of apo A-I to promote cholesterol efflux is partiallydetermined by its ability to bind with lipids. The kinetics of thisbinding was assessed by measuring the rate of DMPC liposome turbidityclearance (FIG. 18). The t_(1/2) values were estimated to be 5.0 and 3.5min, for apo A-I_(unox) and apo A-I_(ox), respectively. Thus, apoA-I_(ox) binds DMPC more rapidly than apo A-I_(unox), although the finalbinding levels are equal. The reduction of apo A-I_(ox) by PMSR in thepresence of DHLA completely restores DMPC binding kinetics (FIG. 18),protein secondary and tertiary structural features, and thermodynamicparameters (Table 1) characteristic of the native apo A-I_(unox). Thus,enzymatic reduction of apo A-I_(ox) using PMSR with DHLA as its cofactorcan reverse the oxidative damage caused by the oxidation of two of threemethionine residues (Met-112 and Met-148).

Well defined rHDL particles with varying proportions of apo A-lox, apoA-I_(unox), and apo A-II, rHDL-1, rHDL-2, rHDL-3, and rHDL-4, werereacted each with PMSR in the presence of DHLA. Conversion oflipid-bound apo A-I_(ox) to apo A-I_(red) proceeded to 50-60% after 5min of incubation independent of rHDL protein composition (FIGS. 19A-E).Rate and yield of the reaction were similar to those observed forlipid-free apo A-I. No changes in size distributions or shapes in rHDLparticles were observed after PMSR reaction as analyzed by nondenaturinggradient gel electrophoresis and electron microscopy.

These data indicate that both of the apo A-I methionine sulfoxideresidues (Met-112 and Met-148) are as accessible to enzymatic reductionin rHDL particles as they are in lipid-free apo A-I_(ox). Furthermore,the reaction does not depend on the molar ratio of apo A-I_(unox)/apoA-I_(ox) or the presence of apo A-II. Because these experiments wereperformed with discoidal rHDL particles including those that contain apoA-II, these findings as it is important for the compositions of thepresent invention, are especially relevant to native nascent HDL, butthey also should apply to the spherical rHDL compositions of the presentinventions in terms of resembling with mature HDL (Brouillette C. G. &Anantharamaiah G. M. Biochim Biophys Acta 1995; 1256:103-29), includingthose containing only apo A-I or both apo A-I and apo A-II.

In certain embodiments of the present invention, it may be desirable toincorporate DHLA into the rHDL compositions of the invention to restoresulfoxidized methionines of targeting apolpoproteins A-I and A-II andfragments thereof at sites of interest (back to methionine native form).

Example 6: Cholesterol Efflux by Native HDL

The ability of native HDL to accept cholesterol from cholesterol-loadedhuman skin flbroblasts was assessed depending on the ratio ofunoxidized/oxidized apo A-I in these particles as described (Sigalov etal. Eur J Clin Chem Clin Biochem 1997; 35:395-6).

HDL-mediated cholesterol efflux was found to depend on the ratio ofnon-oxidized/oxidized apo A-I in HDL (FIG. 20). However, HDL particlescontaining apo A-I_(ox) (with sulfoxidized methionines at positions 112and 148) are still able to promote cholesterol efflux, which isimportant for the compositions of the present invention.

Example 7: Labelling of HDLs with Non-Metallic Atoms

As disclosed in US Pat Appl 20070243136 and incorporated herein byreference, Iodine in all isotopes (¹²³I, ¹²⁵I, ¹²⁷, ¹³¹I) can besuccessfully linked to apo A-I protein using IODO-BEAD Iodinationreagent (Pierce, Rockford, Ill.) in good yield. Once linked, the labeledprotein can be reconstituted with lipids to form the iodinated HDL.Iodinated HDL can be used for PET/SPECT and CT indications. Below is abrief description for protein labeling:

1. Wash beads with 500 ul of reaction buffer per bead. Dry the bead(s)on filter paper.

2. Add bead(s) to a solution of carrier-free ¹²³I, ¹²⁵I, ¹²⁷I ¹³¹I(approximately 1 mCi per 100 ug of protein) diluted with reaction bufferand allow to react for 5 minutes.

3. Dissolve or dilute protein in reaction buffer (TBS) and add to thereaction vessel. Allow the reaction to proceed for 15 minutes.

4. Stop the reaction by removing the solution from the reaction vessel.

5. Dialyze the solution.

For labeling of lipids, the protocol used is similar to the protocolused for labeling lipids with metallic agents. To incorporate ¹⁸Fisotope in HDL, ¹⁸F-fluorodeoxyglucose (FDG a common PET agent) can beintroduced into the core of HDL. Since FDG is neutrally charged itshould easily being incorporated into the core. The material could beproduce as follows:

1. Add apoA-I to the reaction buffer (TBS) that contains a mixture oflipids and FDG.

2. Sonicate.

3. Dialyze the solution.

To attach a positron emitting isotope selected from the group consistingof C¹¹, F¹⁸, O¹⁵, and N¹³, methods described in Miller et al. AngewandteChemie 2008; 47:8998-9033, can be applied by those of ordinary skill inthe art of radiolabeling of proteins, peptides, lipids, and othercompounds. These methods comprise attaching the isotope either directlyby isotopic substitution for an existing atom in the compounds,attachment of an isotope directly to compounds or by attaching aprosthetic group bearing the isotope. The choice of methods forattachment is practiced by those of ordinary skill in the art ofradiolabeling.

Example 8: Derivatization of Phospholipids for Use in rHDL Compositionsof the Invention

As disclosed in US Pat Appl 20070243136 and incorporated herein byreference, the rHDL compositions of the present invention will bedesigned to comprise phospholipids that have been derivatized witheither the metallic ion chelate, the non-metallic imaging agent or, witha targeting components. While the following discussing is based on PEderivatives, it should be understood that any phospholipid, andparticularly, PC, PA, PS, PI, PG, CL and SM could also be derivatized insimilar fashion.

In certain exemplary embodiments of the present invention, PEderivatives for incorporation into rHDL are Gd-DTPA-PE, biotinylated PE,and poly-L-lysine-PE. The purpose of these phospholipid derivatives isto allow specific agents to be incorporated onto the surface of the rHDLcompositions of the invention. Each PE (or other phospholipid)derivative is anchored via its hydrophobic fatty acyl chains into therHDL surface. Each PE derivative projects a functional moiety from therHDL surface, to which the metallic or non-metallic imaging contrastagent may be attached. In specific embodiments, that contrast agent is,for example, Gd (for MR contrast) is iodine or bromine (for CT), ¹¹¹In,⁹⁹mTc (for gamma-scintigraphy), fluorescein (for optical imaging). Inother embodiments, it is contemplated that the rHDL entities of theinvention also will comprise a targeting agent for molecular targetingof the rHDL imaging particles. The targeting agent, e.g., an antibodywill be attached to a PE (or other phospholipid moiety) head groupthrough a functional group. Preferably, the contrast agent and targetingagent are on separate phospholipid molecules within the rHDL.

In specific examples, the metallic paramagnetic ion is Gd, which isattached to the PE covalently linked to DTPA, a chelating agent. Othermetallic agents and chelating agents have been discussed above. Thesynthesis of DTPA-PE is routinely performed by incubating PE with cyclicDTPA anhydride (cDTPAA), followed by column chromatography purification.Gd incorporation is then achieved by treating gadolinium chloridehexahydrate (Aldrich, Milwaukee, Wis.) with DTPA-PE and purifiedGd-DTPA-PE complexes using column chromatography. In more detail, thissynthesis is described in US Pat Appl 20070243136, the disclosure ofwhich is incorporated herein by reference.

To attach antibodies or other targeting agent, biotinylated-PE or otherphospholipid (available from Avanti Polar Lipids) may be used. As withDTPA-PE, the biotinylated PE is substituted for PE during thereconstitution of HDL particles. In exemplary embodiments, the targetingagent is an antibody. The biotin group is exposed at the surface of therHDL, so that antibodies conjugated to avidin and mixed with the rHDLwill self-associate and be available for binding to their targetantigens (e.g., see Lanza et al. Circulation 2002; 1062842-7).

In a particular embodiment, it may be desirable to increase the amountof contrast agent or targeting agent in the rHDL particle, but withoutproducing a concomitant increase the mass of derivatized PE perparticle. This may be necessary in the event that the mole % DPTA-PEthat replaces the PE in the HDL disrupts the structure of the rHDL. Asnoted above, this is highly unlikely because DPTA-PE substitutes wellfor PE into related structures such as liposomes. Nevertheless, afeasible way to substantially lower the content of DPTA-PE in the rHDLis to use an established procedure in which a poly-L-lysine linker isattached to PE, so that at each epsilon-amino group, a DPTA moiety iscovalently attached. In this way, less PE, but more Gd (and/or othermetal), can be incorporated into the rHDL. Thus, it is contemplated thatthe use of the poly-L-lysine-PE will allowing multiple metallic ions tobe chelated. This is a standard method that has previously been used inliposomal applications (e.g., see Torchilin V. P. Adv Drug Deliv Rev2002; 54:235-52). Use of this strategy will facilitate a furtherincrease in the amounts of signal intensity. Similarly, the samepoly-L-lysine-PE can be used to load additional antibody molecules/rHDL(Slinkin et al. Bioconjug Chem 1991; 2:342-8). Thus, it is contemplatedthat a single PE molecule will have multiple metallic contrast agentslinked thereto, and/or multiple targeting agents linked thereto.

Example 9: Production of Lipoprotein Apolipoprotein A-1(LpA-I)-Gadolinium

Complexes.

Gadolinium 1,2-dimyristoyl-sn-glycero-3-phosphoethanolaminediethylenetriamine pentaacetic Acid (GdDMPE-DTPA).

As disclosed in US Pat Appl 20070243136 and incorporated herein byreference, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolaminediethylenetriamine pentaacetic acid (25 mg, 22.8 unol) is heated toreflux with gadolinium triflate (7 mg, 22.8 umol) in dry acetonitrile (4mL) for 18 h. The solution is then allowed to cool and the solvent isevaporated to afford a pale yellow solid that is used without previouspurification.

Preparation ofSpherical LpA-I-Gd Complexes Reconstituted LpA-I/Gdcomplexes are prepared by cosonication of POPC (palmitoyl oleoylphosphatidylcholine), TG (triglyceride, for example, triolein), FC (freecholesterol), C (cholesterol ester), apoA-I and GdDMPEDTPA. Specificamounts of POPC, TG, C, FC and GdDMPEDTPA or other lipophilic gadoliniumcomplexes in chloroform are dried under nitrogen into a 12×75 mm glasstest tube and 800 uL of PBS is added. The lipid-buffer mixture issonicated for 1 min in a 15° C. water bath, incubated for 30 min at 37°C., and sonicated again for 5 min. ApoA-I is subsequently added to thelipid suspension and the protein-lipid mixture is sonicated four times(1 min each time), punctuated by 1-min cooling periods. All LpA-Icomplexes are filtered through a 0.22 um syringe tip filter andreisolated either by size-exclusion chromatography on a Superose 6column or by sequential density gradient ultracentrifugation.

Example 10: Methods of Confirming the Configuration of the rHDL

Having produced the rHDL compositions as described in US Pat Appl20070243136 and incorporated herein by reference, it may be advantageousto determine the mole % of each component of the rHDL compositions ofthe invention. This may be determined by the biochemical assays known tothose of skill in the art (Shamir et al. J Clin Invest 1996;97:1696-704). The Gd content is determined by inductively coupled plasma(ICP) analyses (Gailbraith Laboratories, Inc. (Knoxville, Tenn.)(Fossheim et al. Magn Reson Imaging 1999; 17:83-9; Glogard et al. Int JPharm 2002; 233:131-40), and/or by size exclusion chromatography (gelfiltration) through a calibrated column (e.g., Superose or Sepharosechromatography column).

The penetration of Gd-rHDL into the interstitial space, including theinterior of atherosclerotic plaques, can be affected by particle sizeand to some extent by surface charge. Particle size can be determined bynon-denaturing gel electrophoresis through 2-16% and 4-30% gels, asdescribed (Williams K. J. & Scanu A. M. Biochim Biophys Acta 1986;875:183-94). In addition, the size of the rHDL can be determined usingLaser Light Particle Sizer (Model HPPS 500, Malvern Instruments Inc.,Southborough, Mass.). Alternatively, particle size can be determined byelectron microscopy as described in Example 5. See also Sigalov A. B. &Stern L.

J. Chem Phys Lipids 2001; 113:133-46.

As discussed in US Pat Appl 20070243136 and incorporated herein byreference, it is preferred that the Gd-rHDL should average 5-12 nm indiameter. Surface charge of these moieties may be assessed by agarosegel electrophoresis, as described in previous publications (Sparks etal. J Biol Chem 1992; 267:25839-47; Sparks D. L. & Phillips M. C. JLipid Res 1992; 33:123-30). In all of the above methods, normal humanHDL may be used as reference standard. Substantial deviations of eitherthe size or surface charge from normal HDL could impair particlepenetration into the interstitial space, which may be measured directlyin vivo as described below.

In the event that the surface charge of the rHDL is substantiallydifferent from normal HDL and if this altered surface chargesubstantially decreases penetration of the particles into theinterstitium in vivo, it is contemplated that small amounts ofpositively (basic) or negatively (acidic) charged lipids may be added asneeded to restore a more natural total surface charge. With thismodification, interstitial penetration will be achieved. In the eventthat the size of the rHDL (either with the Gd chelates or with theGd-antibody-chelates) is substantially larger than normal HDL,accompanied by a large decrease in penetration into the interstitum invivo, it is proposed that discoidal reconstituted particles (discussedabove) should be used. This is because variants of the Gd chelates wouldexpand the particles in a direction perpendicular to the surfaces, butnot the edges, of the disks. Thus, the largest overall dimension wouldnot increase. Of note, rHDL disks have also proven useful as drugdelivery vehicles (Rensen et al. Adv Drug Deliv Rev 2001; 47:251-76), sothe technology is reasonably mature. Additionally, in order to reducethe size of the rHDLs comprising antibodies as the targeting moieties,it should be possible and desirable to use antigen-binding fragments ofthe antibody (e.g., Fab or other antibody fragments), rather than thewhole antibody molecules, which would also reduce overall particle size.

Example 11: Use of rHDL in MRI in Mice

As disclosed in US Pat Appl 20070243136 and incorporated herein byreference, model animals can be used to image atherosclerotic plaques,to test the efficacy of the compositions of the present invention. ApoE-KO and Wild Type (WT) mice in the C57B1/6 background weighing 15-40 g,(Jackson Laboratories, Bar Harbour, Me.) are preferably be used. Afterweaning (4 weeks of age) and a 2 weeks period on a regular chow diet,the apoE-KO mice are fed a Western Diet (Research Diets, New Brunswick,N.J.) for up to 42 weeks.

The scheme for in vivo contrast enhanced (CE) MRM in the above mice isdescribed in detail in US Pat Appl 20070243136 and incorporated hereinby reference. Before MRM, an 27-ga. needle is inserted in the tail veinof the mouse and the needle is connected to a saline filled micropolyurethane catheter (Harvard Apparatus, Holliston, Mass.) with aY-site connector, and attached to a syringe pump (Braintree ScientificInc., Braintree, Mass.) while the other connector is attached to asaline-filled syringe for flushing before and after injection of thecontrast agent. The animals are anesthetized with an isoflurane/O₂ gasmixture (4%/400 cc/min initial dose, 1.5%/150 cc/min maintenance dose),which is delivered through a nose cone. The anesthetized animals arethen placed in the RF coil with the animal handling system. An imageintensity standard of 2% agar in 1 mM Gd-DTPA (Magnevist) may be placedbeside the animal for data normalization.

In the exemplary embodiments described in US Pat Appl 20070243136 andincorporated herein by reference, the in vivo MRM is performed with a9.4 T, 89 mm-bore system operating at a proton frequency of 400 MHz(Bruker Instruments, Billerica, Mass.). Constant body temperature of 37°C. is maintained using a thermocouple/heater system. A respiratorysensor can be placed on the abdomen of the animal for monitoring thedepth and frequency of respiration. ECG monitoring will be performedusing subcutaneous silver electrodes. The sensors are connected to thesmall animal monitoring unit.

After anatomical and angiographic MR localization, multi-contrasthigh-resolution images of the wall perpendicular and/or parallel to theentire arterial tree are obtained. MRM imaging is performed with the 2Dand 3D high-resolution MR software and hardware methods describedpreviously (Fayad et al. Circulation 1998; 98:1541-7; Choudhury et al.Atherosclerosis 2002; 162:315-21; Choudhury et al. J Magn Reson Imaging2003; 17:184-9; Itskovich et al. Magn Reson Med 2003; 49:381-5). Withoutremoving the animal and the coil, the contrast agent is administeredusing the syringe pump at a constant rate of 50 ml/min. The procedure isfollowed with flush of saline.

Briefly, in order to characterize the plaque the following MRM isconducted as described in US Pat Appl 20070243136: 1) multicontrastblackblood pre-contrast enhanced (CE) MRM; and 2) post-contrastnon-specific (Gd-DTPA) CE MR black-blood T1W imaging.

Histopathology, image and data analysis are performed to evaluate theMRM imaging with and without contrast-enhanced (Gd-DTPA) techniques. Forhistology processing, following MRM, randomly selected animals aresacrificed. The aorta from these animals is perfused, removed, and fixed(Fayad et al. Circulation 1998; 98:1541-7; Rong et al. Circulation 2001;104:2447-52; Reis et al. J Vasc Surg 2001; 34:541-7; Choudhury et al.Atherosclerosis 2002; 162:315-21; Choudhury et al. J Magn Reson Imaging2003; 17:184-9; Itskovich et al. Magn Reson Med 2003; 49:381-5). Serialsections of the aorta are cut at intervals matching corresponding MRMimages. Co-registration is performed using external landmarks to theaorta, including arterial branches and the image processing algorithmsexplained above. Surrounding tissue is included in the sections forarterial support during fixation and to enhance co-registration throughthe use of fiducial markers as previously reported (Choudhury et al.Atherosclerosis 2002; 162:315-21; Fayad et al. Circulation 1998;98:1541-7; Reis et al. J Vasc Surg 2001; 34:541-7; Choudhury et al. JMagn Reson Imaging 2003; 17:184-9; Itskovich et al. Magn Reson Med 2003;49:381-5). The specimens are embedded in paraffin, and sections 5 umthick are cut and stained with combined Masson's trichrome elastin (CME)stain and hematoxylin and eosin (H&E) stain. Other staining proceduresalso may be used.

Image analysis for plaque morphology (size, volume, etc.) andcharacterization may be performed by applying the cluster analysis,snake-based contour detection, and coregistration algorithms to both theMRM and histopathology images. The effectiveness of cluster and snakeanalyses as methods of automated atherosclerotic plaque componentsegmentation can be validated by comparing the registered colorcomposite MRM images with the corresponding histopathology sections inboth a qualitative and quantitative manner.

Qualitatively, the individual plaque components are identified on thecluster analyzed color composite matched MR images and histologicalslices for every specimen on the basis of signal intensity in themulticontrast MR images and histopathological staining. The colorcomposite MR images and histopathological images are rated according tothe histopathological classification from the Committee on VascularLesions of the Council of Atherosclerosis of the American HeartAssociation (AHA) (Fayad et al. Circulation 1998; 98:1541-7; Fayad etal. Circulation 2000; 101:2503-9; Stary et al. Circulation 1995;92:1355-74) which have been used in US Pat Appl 20070243136. See alsoChoudhury et al. Atherosclerosis 2002; 162:315-21; Choudhury et al. JMagn Reson Imaging 2003; 17:184-9; Helft et al. Circulation 2002;105:993-8).

Quantitatively, the digitized histopathological slices are subjected tothe same cluster analysis procedure that the corresponding colorcomposite MRM images were subjected to. The clustered histopathologicalimages are characterized according to the AHA classification as above.Using the cluster and snake-based algorithms, the areas of these labeledregions are computed for both datasets as a percentage of total vesselwall area. The histopathological slices are matched to theircorresponding MRM slices to allow for direct comparison of the labeledregion areas. Additionally, The signal intensity of the vessel wallcomponents and the adjacent muscle and the image intensity standard (todefine the background MR signal) over time are determined by means ofstandard region-of-interest (ROI) measurements on the corresponding MRMimages. An ROI placed outside the body, that contained no motionartifacts, will be selected to measure the standard deviation of thenoise signal. Both normalized signal intensity (SI):SI=SI_(plaque-post)/SI_(plaque-pre) and contrast-to-noise ratios (CNR):CNR=SI_(plaque)−SI_(muscle)/SD_(noise) can be calculated.

For histopathological analysis of plaques, the components of thearterial wall and the atherosclerotic plaque can be determined bymethods previously described in US Pat Appl 20070243136. See also Fayadet al. Circulation 1998; 98:1541-7; Rong et al. Circulation 2001;104:2447-52. Three categories of plaque components may be correlatedwith the MRI images: 1) fibrous, 2) lipid, and 3) calcium. Thesecategories are identified histopathologically by the following methods:intense green staining by CME (fibrous); foam cells and cholesterolclefts on H&E and CME (lipids); acellular purple crystals by H&E(calcium). In addition, immunohistochemical staining can be performedfor □-actin (smooth-muscle cells), CD68 and MOMA2 (macrophages), the twoprincipal cell types found in plaques.

After both MR images and histological sections are reviewed andcategorized, comparison between the two sets of data can be performed.Given the difference in slice thickness between MRM histologicalcross-sections, three to four histological sections for each MR imagelocation should be selected based on the relative distance of the MRMand histological sections from renal arteries and iliac bifurcation. Inorder to correct for shrinkage of the aortic specimen duringhistological processing, additional measures other than distance fromthe bifurcation for matching of the MRI and histological sections can beused. For example, the gross morphological features of the lumen andvessel wall, such as, the overall shape and size of the lumen andwall—may be compared. In addition, the location of large calcifiedregions, which appear hypointense on MRM, will aid in matching thecross-sections at each location. An agreement between MRI and histologymay be defined as the presence of any plaque component region in thesame quadrant on the MRI section and in all 3-4 of the matchedhistological sections. Pre-CE and post-MRM should be matched andregistered.

Example 12: Use of Modified HDL as Specific Carrier for MRI ofAtherosclerotic Plaques

As discussed herein and in US Pat Appl 20070243136, the disclosure ofwhich is incorporated herein by reference, the ability to image thepresence or biological activity of specific molecules in vivo (i.e.,molecular imaging) in atherosclerotic plaques is of considerableinterest. Current non-contrast and contrast-enhanced methods do notinterrogate specific biochemical processes. In certain embodiments, thepresent invention is designed to enhance distinctions among plaquecomponents by the introduction of plaque-specific contrast agents thatare related to molecular signatures involved in atherosclerosis. Asdiscussed herein throughout, synthetic nanoparticles that mimic HDL areeasily reconstituted, can carry a considerable contrast agent (i.e., Gd)payload, and are sufficiently small to penetrate readily in theextracellular space and freely enter and exist plaques. The presentexample describes methods and results of the use of nanoparticlescontaining a lipophilic gadolinium complex to create an MR contrastagent and the use of the nanoparticles as as a diagnostic marker foratherosclerotic disease.

As disclosed in US Pat Appl 20070243136, and incorporated herein byreference, apoA-I/POPC/GdDTPADMPE/sodium cholate rHDL can be prepared byspontaneous association of lipid-free apoA-I and small unilamellarvesicles of POPC (palmitoyloleoyl phosphatidylcholine), and GdDTPEDMPE(dimiristoyl phosphatidylethanolamine). POPC (2.4 mg) and GdDTPADMPE(0.4 mg) in chloroform are dried in a thin film under nitrogen. Sodiumcholate (3.1 mg) dissolved in TBS (200 uL) is added to the lipid film togive a turbid suspension that clarified after incubation at 37° C. for1.5 hours. To the clear solution is added apo A-I (1 mg) dissolved in 1mL of TBS and the resulting mixture is allowed to incubate for one hourat 37° C. (Clay et al. Atherosclerosis 2001; 157:23-9). After incubationthe sample is exhaustively dialyzed to get rid off the excess ofcholate. The rHDL-GdDTPA-DMPE contrast agent diameter is determined witha laser light-scattering submicron particle sizer. Thirteen-month-oldatherosclerotic apolipoprotein E knockout (KO) mice (n=4) on a high fatdiet and Wild Type (WT) (n=4) group undergo in vivo MR microscopy (MRM)of the abdominal aorta using a 9.4T MR system. Pre- and post-contrastenhanced (CE) (1 hour post) MRM is performed using a T1W black bloodsequence. Sixteen contiguous 500 um thick slices with an in-planeresolution of 93 um are acquired in 30 minutes. The rHDL contrast agent(47.2 nmol) is injected via the tail vein. MRM images of the matched(pre and post) slices are used for analysis.

The diameter of the rHDL contrast agent is 47 nm. The in vivo MR imagesreveal that after 1 hour post-injection of rHDL-GdDTPA-DMPE asubstantial enhancement in the plaque in the abdominal aorta isobserved. The ratio of the post to pre signal intensity of wallnormalized with respect to muscle is 1.21 (21% enhancement) in KO miceafter 1 hour (FIG. 5). There was no enhancement in the WT group.

These data demonstrate that in this in vivo MR study, Gd loadednanoparticles localize and substantially enhance imaging ofatherosclerotic plaques. As disclosed in the present invention,targeting molecules such as modified apo A-I and A-II and fragmentsthereof can be easily incorporated in the rHDL-GdDTPA-DMPE contrastagent. The targeting moieties of the present invention facilitatedelivery and retention of the nanoparticles containing the contrastagent into plaques. This provides a way to achieve noninvasive optimalsensitive and specific in vivo molecular detection of atherosclerosisusing MR.

Example 13: Discussion of Selection of Contrast Agents for MRI

As discussed herein and in US Pat Appl 20070243136, the disclosure ofwhich is incorporated herein by reference, contrast agents are used invarious imaging modalities to enhance tissue contrast or to provide anindication of organ function or flow. One of the major differencesbetween MRI and other imaging modalities is that in MRI the alterationsin signal intensities of tissue depends on the effects of the contrastagents on the MR properties (i.e., water relaxation rates) rather thandirect visualization of the contrast agent itself. Therefore, MRIcontrast agents are imaged indirectly, by their effect on waterrelaxation rates. The present application focuses primarily on agentsthat are injected into the body and use chelated metals to change thewater relaxation properties.

Many metal ions are good candidates as MR contrast agents. Paramagneticcontrast agents (atoms or molecules that have electrons in unpairedstates) are the most commonly used today. Paramagnetic substances caninfluence relaxation rates in two distinctly different but relatedways: 1) through alterations in the local magnetic fields by changingthe local magnetic susceptibility; and 2) through an electron-nucleardipolar interaction. The paramagnetic metal ion used in most currentcardiovascular applications is gadolinium (Gd³⁺) or Gd.

Metallic contrast agent properties are often discussed in terms ofrelaxivity. Since water is present at a very high concentration (55000mM) and the contrast agent is typically at much lower concentration(0.1-1 mM) the contrast agent must act catalytically to relax the waterprotons to have a measurable effect. The relaxivities, r₁ and r₂, thusdescribes this catalytic efficiency.

In recent years, the Food and Drug Administration has approved a numberof contrast agents for human use: Magnevist® (gadopentetate dimeglumine;Bayer Shering Pharma), Dotarem® (gadoterate meglumine; Guerbet,Aulnay-sous-bois, France), Omniscan® (gadodiamide; Nycomed, Oslo,Norway), ProHance® (gadoteridol; Bracco SpA, Milan, Italy), Gadovist®(gadobutrol; Bayer Shering Pharma), MultiHance® (gadobenate dimeglumine;Bracco SpA), OptiMARK® (gadoversetamide; Mallinkrodt, St. Louis, USA),Primovist® (gadoxetic acid; Bayer Shering Pharma) in Europe, or Eovist®in USA, and Vasovist® (gadofosveset trisodium; Epix Pharmaceuticals,Cambridge, USA). The methods and compositions of the present inventionmay use any one or more of these approved metallic agents.

The majority of contrast agents used are gadolinium based. Other metalsare capable of providing contrast, and iron and manganese have been usedin commercially approved agents. In addition to the agents listed above,other contrast agents that may be used include MS-325 (EPIX MedicalInc), B22956 (Bracco Diagnostics), both GD-based contrast agents thatreversibly bind serum albumin. Blood pooling agents such as Gadomer-17(Schering AG), and P792 (Laboratoire Guerbet), and Iron oxide particlese.g., AMI-25, AMI-227 (Advanced Magnetics); NC100150 (Nycomed) also maybe used in the applications described herein.

Gadolinium chelates are generally administered intravenously althoughsome oral preparations have been described. Where intravenousadministration is performed, it is often recommended that injection ofGd be followed by an injection of saline flush. The effective dose of Gdis approximately 0.1 mmol/kg of body weight (0.2 cc/kg or 0.1 cc/lb).However, the FDA has approved gadoteridol for triple dose injectionvolumes where an initial dose of 0.1 mmol/kg is followed by 0.2 mmol/kgup to 30 minutes after the initial dose. Such FDA approved protocols maybe used to provide general guidance as to amounts and regimens in whichthe synthetic nanoparticle compositions of the present invention shouldbe administered to a subject for imaging purposes.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

Example 14: Use of Modified Apolipoprotein-Liposomes for CT

As discussed herein and in U.S. Pat. No. 6,248,353, the disclosure ofwhich is incorporated herein by reference, proteins can be incorporatedinto liposomes by incubating the protein solution with the solution ofpreformed liposomes. The present example describes methods and resultsof the use of nanoparticles containing an iodinated contrast agent tocreate a CT contrast agent and the use of the nanoparticles as adiagnostic marker for CT-based vascular imaging and detection andlocalization of neoplastic and inflammatory lesions (see e.g., Mukundanet al. AJR Am J Roentgenol 2006; 186:300-7; Zheng et al. Contrast MediaMol Imaging 2010; 5:147-54).

As described in Mukundan et al. AJR Am J Roentgenol 2006; 186:300-7 andKao et al. Acad Radiol 2003; 10:475-83, and incorporated herein byreference, liposomal formulations containing an iodinated contrast agentsuch as Iodixanol or Iohexol can be prepared by encapsulation of aniodinated contrast agent into the liposomes. Iodixanol (Visipaque 320,GE Healthcare) is concentrated using a FreeZone 4.5-L Benchtop FreezeDry System (Labconco). A lipid mixture (200 mmol/L) consisting of1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol, and1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethyleneglycol)-2000] (DSPE-MPEG 2000) in a 55:40:5 molar ratio is dissolved inethanol at 70° C. The ethanol solution is then hydrated withconcentrated Iodixanol (480 mg I/mL) for 2 hr. Liposomes are extrudedwith a 10-mL Lipex Thermoline extruder (Northern Lipids) with fivepasses through a 0.2-μm Nuclepore membrane (Waterman) and seven passesthrough a 0.1-μm Nuclepore membrane (Waterman). Liposomes are thendialyzed overnight in a 100,000-molecular weight cutoff (MWCO) dialysisbag against phosphate-buffered saline to remove ethanol and freeiodixanol. The resulting liposomal iodixanol formulations (43.8 mg I/mL)are then concentrated using a Pellicon tangential flow filtrationcassette and Labscale TFF system (Millipore) to a final concentration of118.6 mg 1/mL and stored in phosphate-buffered saline at pH 7.2. Thesize of the resultant liposomal formulations obtained is determined bydynamic light scattering (DLS) using a BI-9000AT Digital Autocorrelator(Brookhaven Instruments), a BI-200SM goniometer (JDS Uniphase), and aHamamatsu photomultiplier (Brookhaven). The iodine concentrations of theliposomal formulations are determined by measuring the absorption ofultraviolet (UV) light at 246 nm with a UV-visible lightspectrophotometer. C57BL/6 mice are used for animal studies. Iodinatedliposomes are infused via a tail vein cannula at a volume dose of 0.5mL/25 g of mouse weight. The micro-CT system is used to acquire CTimages (Badea et al. Med Phys 2004; 31:3324-9; Mukundan et al. AJR Am JRoentgenol 2006; 186:300-7; Zheng et al. Mol Pharm 2009; 6:571-80).

The in vivo CT images reveal that the liposomal-based iodinated contrastagent shows long residence time in the blood pool, very high attenuationwithin submillimeter vessels, and no significant renal clearancerendering it an effective contrast agent for vascular imaging. Theseresults (Mukundan et al. AJR Am J Roentgenol 2006; 186:300-7) and databy others (Zheng et al. Contrast Media Mol Imaging 2010; 5:147-54)demonstrate that an iodinated contrast agent-loaded nanoparticleslocalize and substantially enhance CT imaging in vivo.

As an alternative embodiment, a vesicle-forming iodinated contrastagent,1-palmitoyl-2-((E)-10,11-diiodo-undec-10-enoyl)-sn-glycero-3-phosphocholine,for applications in CT imaging can be prepared by chemical modificationof a phosphatidylcholine lipid that is commonly used in liposomeformation to create an iodinated lipid that self-assembles into about50-150 nm iodoliposomes possessing as-prepared imaging contrastfunctionality as described in (Elrod et al. Nanomedicine: Nanotech, Bioland Med 2009; 5:42-5). A solution of 10-undecynoic acid (600 mg, 3.29mmol) is prepared by suspending the solid in 10 mL of water followed byaddition of NaOH (135 mg, 3.38 mmol). Subsequently, KI (1.66 g, 10 mmol)is added to the solution, and the reaction mixture is cooled in an icebath. Half of a 14% NaOCl solution (1.77 g, 3.3 mmol) is then addeddropwise with stirring. The reaction is allowed to proceed for 30minutes, after which half of a 50% H2SO4 solution (1.77 g, 9.0 mmol) isadded dropwise with stirring. The remaining portions of the NaOCl andH2SO4 solutions are then added simultaneously. The mixture is removedfrom the ice bath and stirred overnight at room temperature (23°-25°C.). A yellowish-to-tan solid precipitated, is filtered, and is furtherpurified by flash chromatography on silica (4:1 hexanes/ethyl acetate to1:1 hexanes/ethyl acetate). The white solid product is recrystallizedfrom 80% methanol, yielding 586 mg (41% yield). A mixture containing(E)-10,11-diiodo-undec-10-enoic acid (231 mg, 0.53 mmol),N,N′-dicyclohexylcarbodiimide (110 mg, 0.53 mmol),1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (100 mg, 0.20 mmol)and 4-(dimethylamino) pyridine (66 mg, 0.53 mmol) is dissolved in 2 mLof anhydrous chloroform under nitrogen and stirred for 48 hours. Theresulting white suspension is filtered and the filtrate is evaporated.The crude solid is purified by flash chromatography on silica (60:30:5CH2Cl2/methanol/water). The product is identified by thin-layerchromatography (silica) using molybdate staining for visualization.

As discussed in the present invention and in U.S. Pat. No. 6,248,353,the disclosure of which is incorporated herein by reference, targetingmolecules such as modified apo A-I and A-II and fragments thereof can beeasily incorporated in the liposome iodinated contrast agent byincubating the protein or peptide solution with the solution ofpreformed liposomes. The targeting moieties of the present inventionfacilitate delivery and retention of the nanoparticles containing thecontrast agent to macrophage-rich sites of interest, including but notlimiting to, tumor sites and vulnerable atherosclerotic plaques. Thisprovides a way to achieve noninvasive optimal sensitive and specific invivo molecular detection and localization of neoplastic and inflammatorylesions using CT.

Example 15: Use of Modified Apolipoprotein-DOTAP Liposomes for Imaging

As discussed herein and in WO 88/09165, U.S. Pat. Nos. 7,288,266,7,588,751, 6,139,819, 5,676,928, and 5,965,542, US Pat Appls20070065432, 20060204566, 20090012025, 20080286353, 20090311191, and20090312402, the disclosure of which is incorporated herein byreference, highly efficient charged liposomes can be used as an improveddelivery system for biologically-active compounds and contrast agents(see also Templeton N. S. World J Surg 2009; 33:685-97; Gjetting et al.Int J Nanomed 2010; 5:371-83; Oliver et al. Org Biomol Chem, 2006;4:3489-97; Kim et al. J Hepatol 2009; 50:479-88; Leander et al. EurRadiol 2001; 11:698-704; Zheng et al. Contrast Media Mol Imaging 2010;5:147-54; Strieth et al. Clin Cancer Res 2008; 14:4603-11). Toencapsulate therapeutic agents including, but not limiting to,antioxidants i.e. for example, as lipoic and dihydrolipoic acid),anticancer and anti-inflammatory therapeutics (Sigalov A. B. & Stern L.J. Antioxid Redox Signal 2002; 4:553-7; Bharali D. J. & Mousa S. A.Pharmacol Ther 2010; 128:324-35; Biewenga et al. Gen Pharmacol 1997;29:315-31; Bitler B. G. & Schroeder J. A. Recent Pat Anticancer DrugDiscov 2010; 5:99-108; Cuzick et al. Lancet Oncol 2009; 10:501-7; Dhikavet al. JIACM 2002; 3:332-8; Dinarello C A. Cell 2010; 140:935-50; Fu P.& Birukov K. G. Transl Res 2009; 153:166-76; Ghibu et al. J CardiovascPharmacol 2009; 54:391-8; Maczurek et al. Adv Drug Deliv Rev 2008;60:1463-70; Manda et al. Curr Chem Biol 2009; 3:342-66; Packer et al.Free Radic Biol Med 1995; 19:227-50; Rothschild et al. Clin Lung Cancer2010; 11:238-42; Salinthone et al. Endocr Metab Immune Disord DrugTargets 2008; 8:132-42; Souto et al. Curr Eye Res 2010; 35:537-52; Tanget al. Chin J Cancer 2010; 29:775-80; Teicher B. A. & Andrews P. A.,eds. Anticancer drug development guide. Second edition. Totowa, N.J.:Humnana Press; 2004:430), methods described herein and in (WO 88/09165,U.S. Pat. Nos. 7,288,266, 7,588,751, 6,139,819, 5,676,928, and5,965,542, US Pat Appls 20070065432, 20060204566, 20090012025,20080286353, 20090311191, and 20090312402; Templeton N. S. World J Surg2009; 33:685-97; Gjetting et al. Int J Nanomed 2010; 5:371-83; Oliver etal. Org Biomol Chem, 2006; 4:3489-97; Kim et al. J Hepatol 2009;50:479-88; Leander et al. Eur Radiol 2001; 11:698-704; Zheng et al.Contrast Media Mol Imaging 2010; 5:147-54; Strieth et al. Clin CancerRes 2008; 14:4603-11; Souto et al. Curr Eye Res 2010; 35:537-52; BharaliD. J. & Mousa S. A. Pharmacol Ther 2010; 128:324-35) can be applied bythose of ordinary skill in the art of drug delivery, liposomeformulations and lipoproteins. These methods comprise attaching thetherapeutics to nanoparticle by adsorption, incorporation, covalentbonding, chelating, and encapsulation. The choice of methods forattachment is practiced by those of ordinary skill in the art of drugdelivery and formulations.

As described in U.S. Pat. No. 5,676,928 and US Pat Appl 20090312402, andincorporated herein by reference, an autoclaved diagnostic liposomalcomposition comprising a neutral phospholipid and a charged phospholipidand containing at least one X-ray or MRI contrast agent foradministration to human or animal subjects can be prepared. 0.640 g ofhydrogenated phosphatidylcholine derived from egg yolk (HEPC), 0.064 gof hydrogenated phosphatidylserine (HEPS) synthesized from HEPC, and 60ml of a mixture of chloroform, methanol and water (volume ratio100:20:0.1) are mixed in a flask. The mixture is heated on a water bath(65° C.) to dissolve the phospholipids, and the resulting solution isheated in a rotary evaporator at 60° C. to evaporate the solvent. Theresidue is further dried in vacuum for 2 hours to form a lipid film. Anaqueous solution containing iodixanol (1,3-bis(acetylamino)-N,N′-bis3,5-bis(2,3-dihydroxypropylaminocarbonyl)-2,4,6-triiodophenyl!-2-hydroxypropane)(0.4 g/ml) and sucrose (0.05 g/ml) is heated to 65° C., and 10 ml of theheated solution is combined with the lipid film, and the mixture isstirred with a mixer for 10 minutes while heating at 65° C. This mixtureis filtered once under pressure through a polycarbonate membrane filterhaving a pore size of 1.0 μm to yield multilamellar vesicles of therequired size (MLV). The liposome suspension obtained is sterilized anddiluted with isotonic glucose to a concentration of 50 mg encapsulatediodine/ml. The composition is then injected intravenously into ratscarrying multiple hepatic cancer metastases. At doses of 50 and 100 mgencapsulated iodine/kg, X-ray attenuations of 42 and 62 HU respectivelyare observed in the normal regions of the liver, while attentuation intumor metastases are minimally affected. Macroscopic analysis shows thatdetected tumors are smaller than 5 mm in diameter. These resultsdemonstrate that hydrophilic compounds such as iodinated contrast agentscan be encapsulated into anionic liposomes and i.v. administered tolocalize and substantially enhance CT imaging in vivo.

As described in Strieth et al. Clin Cancer Res 2008; 14:4603-11 andincorporated herein by reference, cationic liposomes can be used forencapsulation of hydrophilic compounds such as Paclitaxel. Cationicliposomes with a total lipid content of 20 mmol/L are prepared by thelipid film method followed by several cycles of extrusion. Forpaclitaxel containing liposomes (EndoTAG-1), 0.1 mmol DOTAP, 0.094 mmolDOPC, and 0.006 mmol paclitaxel are dissolved in 15 mL chloroform(Merck). For control experiments, cationic liposomes without paclitaxel(CL) are prepared by dissolving 0.1 mmol DOTAP and 0.1 mmol DOPC in 15mL chloroform. For fluorescence microscopy experiments, 0.05 mmol DOTAP,0.046 mmol DOPC, and 0.004 mmol Rh-DOPE are dissolved in 15 mLchloroform. The particle size of the liposomes was analyzed by photoncorrelation spectroscopy using a Malvern Zetasizer 3000 (MalvernInstruments). Using dorsal skinfold chamber preparations in SyrianGolden hamsters, in vivo fluorescence microscopy experiments are doneafter repeated a liposome-encapsulated paclitaxel treatment of A-Mel-3tumors. Controls receive glucose, paclitaxel alone, or cationicliposomes devoid of paclitaxel. Extravasation of rhodamine-labeledalbumin is measured to calculate microvessel permeability, andintratumoral leukocyte-endothelial cell interactions are quantified.Subcutaneous tumor growth is evaluated after combination therapyfollowed by histologic analysis. Microvascular permeability wassignificantly increased only after treatment with aliposome-encapsulated paclitaxel, whereas intratumoralleukocyte-endothelial cell interactions are not affected by anytreatment. In separate skinfold chamber experiments, fluorescentlylabeled cationic liposomes keep their targeting property for tumorendothelial cells after repeated a liposome-encapsulated paclitaxeltreatment and no signs of extravasation are observed. SubcutaneousA-Mel-3 tumor growth is significantly inhibited by the combination ofcisplatin and a liposome-encapsulated paclitaxel. These resultsdemonstrate that hydrophilic compounds such as iodinated contrast agentscan be encapsulated into cationic liposomes and i.v. administered.

As described in Kim et al. J Hepatol 2009; 50:479-88 and incorporatedherein by reference, DOTAP/cholesterol/apo A-I compositions can be usedfor encapsulation and delivery of hydrophilic compounds such as siRNA.Cationic liposomes are prepared by mixing1,2-dioleoyl-3-trimethylammonium-propane (DOTAP; Avanti Polar Lipids,Alabaster, Ala.) and cholesterol (Sigma, St. Louis, Mo.) in aDOTAP:cholesterol ratio of 1:1 (mol:mol). To formulate apo A-I-boundliposomes, cationic liposomes are incubated with apo A-I at alipid/protein ratio of 10:1 (w/w) overnight at 4° C. The incorporationyield of apo A-I into liposomes is examined by labeling of the lipidcomponent with lissamine rhodamine B-diacyl phosphatidylethanolamine(Rho-PE; Avanti Polar Lipids). The labeled self-assembledRho-PE/DOTAP/cholesterol/apo A-I liposomes are loaded onto a sepharoseCL-4B column (80 cm×1.6 cm; Pierce, Rockford, Ill.), and bothfluorescence intensity and protein concentration are measured in eachfraction. DOTAP/cholesterol/apo A-I are used for encapsulation ofhydrophilic siRNA and intravenously administered in mice with hepatitisC virus to assess antiviral activity as well as the duration ofsilencing. The results suggest that DOTAP/cholesterol/apo A-I liposomeis a highly potential delivery vehicle to transfer hydrophiliccompounds.

These results of in vitro and in vivo studies demonstrate that asdescribed in the present invention and in U.S. Pat. No. 6,248,353 andKim et al. J Hepatol 2009; 50:479-88, targeting molecules such asmodified apo A-I and A-II and fragments thereof can be easilyincorporated in the liposome contrast agent formulations by incubatingthe protein or peptide solution with the solution of preformed liposomesor by preparation of liposomes in the presence of the protein or peptidemolecules of the present invention. The targeting moieties of thepresent invention facilitate delivery and retention of the nanoparticlescontaining the contrast agent to macrophage-rich sites of interest,including but not limiting to, tumor sites and vulnerableatherosclerotic plaques. This provides a way to achieve noninvasiveoptimal sensitive and specific in vivo molecular detection andlocalization of neoplastic and inflammatory lesions using imagingtechniques such as computed tomography (CT), gamma-scintigraphy,positron emission tomography (PET), single photon emission computedtomography (SPECT), magnetic resonance imaging (MRI), and combinedimaging techniques.

INCORPORATION BY REFERENCE

The references cited herein throughout, to the extent that they provideexemplary procedural or other details supplementary to those set forthherein, are all specifically incorporated herein by reference.

All of the patents and publications cited herein are hereby incorporatedby reference. Each of the applications and patents cited in this text,as well as each document or reference cited in each of the applicationsand patents (including during the prosecution of each issued patent;“application cited documents”), and each of the PCT and foreignapplications or patents corresponding to and/or paragraphing priorityfrom any of these applications and patents, and each of the documentscited or referenced in each of the application cited documents, arehereby expressly incorporated herein by reference. More generally,documents or references are cited in this text, either in a ReferenceList, or in the text itself; and, each of these documents or references(“herein-cited references”), as well as each document or reference citedin each of the herein-cited references (including any manufacturer'sspecifications, instructions, etc.), is hereby expressly incorporatedherein by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method, comprising: a) providing; i) a subject comprising a targetsite to be imaged; ii) a synthetic reconstituted lipoproteinnanoparticle, comprising: a discoidal synthetic phospholipid layer; atleast one synthetic apolipoprotein, wherein said at least one of saidsynthetic apolipoprotein comprises at least one oxidized amino acidresidue selected from the group consisting of tyrosine, methionine andphenylalanine; and at least one contrast agent; wherein said syntheticreconstituted lipoprotein nanoparticle lacks core lipids and onlyprotein constituents are oxidized. b) administering said syntheticreconstituted lipoprotein nanoparticle to said subject under conditionssuch that an image of said target site is created.
 2. The method ofclaim 1, wherein said target site is selected from the group consistingof an atherosclerotic plaque and a tumor.
 3. The method of claim 1,wherein said at least one oxidized amino acid residue comprises amodification selected from the group consisting of hydroxylation,peroxidation, dimerization, sulfoxidation, nitration and halogenation.4. The method of claim 1, wherein said synthetic apolipoprotein isselected from the group consisting of a modified apolipoprotein A-I, amodified apolipoprotein A-II, a modified apolipoprotein A-IV, a modifiedapolipoprotein B, a modified apolipoprotein C-I, a modifiedapolipoprotein C-II, a modified apolipoprotein C-III, and a modifiedapolipoprotein E.
 5. The method of claim 1, wherein said syntheticapolipoprotein is an amphipathic apolipoprotein or a fragment thereof.6. The method of claim 1, wherein said contrast agent is selected fromthe group consisting of Gd(III), Mn(II), Mn(III), Cr(II), Cr(III),Cu(II), Fe (III), Pr(III), Nd(III) Sm(III), Tb(III), Yb(III) Dy(III),Ho(III), Eu(II), Eu(III), and Er(III), Tl²⁰¹, K⁴², In¹¹¹, Fe.⁵⁹,Tc^(99m), Cr⁵¹, Ga⁶⁷, Ga⁶⁸, Cu⁶⁴, Rb⁸², Mo⁹⁹, Dy¹⁶⁵.
 7. The method ofclaim 1, wherein said contrast agent is selected from the groupconsisting of Fluorescein, Carboxyfluorescein, Calcein, F¹⁸, Xe¹³³,I¹²⁵, I¹³¹, I¹²³, P³², C¹¹, N¹³, O¹⁵, Br⁷⁶, Kr⁸¹, Diatrizoate,Metrizoate, Isopaque, Ioxaglate, Iopamidol, Iohexol, Iodixanol.
 8. Themethod of claim 5, wherein said amphipathic apolipoprotein comprises atleast one constituent selected from the group consisting ofphospholipids, glycolipids and steroids.
 9. The method of claim 1,wherein said contrast agent is conjugated to said phospholipid layer.10. The method of claim 1, wherein said contrast agent is conjugated toan amino acid residue of said apolipoprotein.
 11. The method of claim 1,further comprising two or more different contrast agents.
 12. The methodof claim 1, wherein said composition further comprising a therapeuticagent.
 13. The method of claim 12, wherein said therapeutic agent isselected from the group consisting of lipoic acid, dihydrolipoic acid,antioxidants, anticancer and anti-inflammatory agents.
 14. The method ofclaim 1, further comprising a targeting moiety.
 15. The method of claim14, wherein said targeting moiety is selected from the group consistingof an antibody, a receptor, a ligand, a peptidomimetic agent, anaptamer, a polysaccharide, a drug and a phage display product.
 16. Themethod of claim 15, wherein said antibody is selected from the groupconsisting of antibodies against lipoprotein lipase, oxidized epitopeson atherosclerotic plaques oxLDL MDA, matrix metalloproteinases andanti-tissue factors.
 17. The method of claim 14, wherein said targetingmoiety comprises a detectable label.
 18. The method of claim 1, whereinsaid composition is a pharmaceutical composition comprising apharmaceutically acceptable carrier.
 19. The method of claim 1, whereinsaid nanoparticle has a diameter ranging between approximately 5-25nanometers.
 20. The method of claim 1, wherein said phospholipid layerfurther comprises a chelating agent.
 21. The method of claim 1, whereinsaid contrast agent is attached to said nanoparticle by an associationselected from the group consisting of adsorption, incorporation,covalent bonding, chelation, and encapsulation.
 22. The method of claim1, wherein at least one of said synthetic apolipoproteins comprises amodified phospholipid.
 23. The method of claim 22, wherein said modifiedphospholipid is selected from the group consisting of phospholipidperoxides, dimyristoyl-phosphatidylethanolamine, poly-lysinephosphatidylethanolamine, poly-lysinedimyristoyl-phosphatidylethanolamine
 24. The method of claim 1, whereinsaid synthetic unoxidized phospholipid layer comprises an amphipathiclipid.
 25. The method of claim 24, wherein said amphipathic lipid ispalmitoyl oleoyl phosphatidylcholine.